Sample records for peptide mapping performance
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PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.
Chen, Wenhan; Guo, William W; Huang, Yanxin; Ma, Zhiqiang
2012-01-01
Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/
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NASA Astrophysics Data System (ADS)
Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi
2014-06-01
Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
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Teo, Guoshou; Kim, Sinae; Tsou, Chih-Chiang; Collins, Ben; Gingras, Anne-Claude; Nesvizhskii, Alexey I; Choi, Hyungwon
2015-11-03
Data independent acquisition (DIA) mass spectrometry is an emerging technique that offers more complete detection and quantification of peptides and proteins across multiple samples. DIA allows fragment-level quantification, which can be considered as repeated measurements of the abundance of the corresponding peptides and proteins in the downstream statistical analysis. However, few statistical approaches are available for aggregating these complex fragment-level data into peptide- or protein-level statistical summaries. In this work, we describe a software package, mapDIA, for statistical analysis of differential protein expression using DIA fragment-level intensities. The workflow consists of three major steps: intensity normalization, peptide/fragment selection, and statistical analysis. First, mapDIA offers normalization of fragment-level intensities by total intensity sums as well as a novel alternative normalization by local intensity sums in retention time space. Second, mapDIA removes outlier observations and selects peptides/fragments that preserve the major quantitative patterns across all samples for each protein. Last, using the selected fragments and peptides, mapDIA performs model-based statistical significance analysis of protein-level differential expression between specified groups of samples. Using a comprehensive set of simulation datasets, we show that mapDIA detects differentially expressed proteins with accurate control of the false discovery rates. We also describe the analysis procedure in detail using two recently published DIA datasets generated for 14-3-3β dynamic interaction network and prostate cancer glycoproteome. The software was written in C++ language and the source code is available for free through SourceForge website http://sourceforge.net/projects/mapdia/.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Carr, J. K.; Zabuga, A. V.; Roy, S.; Rizzo, T. R.; Skinner, J. L.
2014-06-01
The spectroscopy of amide I vibrations has become a powerful tool for exploring protein structure and dynamics. To help with spectral interpretation, it is often useful to perform molecular dynamics (MD) simulations. To connect spectroscopic experiments to simulations in an efficient manner, several researchers have proposed "maps," which relate observables in classical MD simulations to quantum spectroscopic variables. It can be difficult to discern whether errors in the theoretical results (compared to experiment) arise from inaccuracies in the MD trajectories or in the maps themselves. In this work, we evaluate spectroscopic maps independently from MD simulations by comparing experimental and theoretical spectra for a single conformation of the α-helical model peptide Ac-Phe-(Ala)5-Lys-H+ in the gas phase. Conformation-specific experimental spectra are obtained for the unlabeled peptide and for several singly and doubly 13C-labeled variants using infrared-ultraviolet double-resonance spectroscopy, and these spectra are found to be well-modeled by density functional theory (DFT) calculations at the B3LYP/6-31G** level. We then compare DFT results for the deuterated and 13C18O-labeled peptide with those from spectroscopic maps developed and used previously by the Skinner group. We find that the maps are typically accurate to within a few cm-1 for both frequencies and couplings, having larger errors only for the frequencies of terminal amides.
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Carr, J. K.; Zabuga, A. V.; Roy, S.; Rizzo, T. R.; Skinner, J. L.
2014-01-01
The spectroscopy of amide I vibrations has become a powerful tool for exploring protein structure and dynamics. To help with spectral interpretation, it is often useful to perform molecular dynamics (MD) simulations. To connect spectroscopic experiments to simulations in an efficient manner, several researchers have proposed “maps,” which relate observables in classical MD simulations to quantum spectroscopic variables. It can be difficult to discern whether errors in the theoretical results (compared to experiment) arise from inaccuracies in the MD trajectories or in the maps themselves. In this work, we evaluate spectroscopic maps independently from MD simulations by comparing experimental and theoretical spectra for a single conformation of the α-helical model peptide Ac-Phe-(Ala)5-Lys-H+ in the gas phase. Conformation-specific experimental spectra are obtained for the unlabeled peptide and for several singly and doubly 13C-labeled variants using infrared-ultraviolet double-resonance spectroscopy, and these spectra are found to be well-modeled by density functional theory (DFT) calculations at the B3LYP/6-31G** level. We then compare DFT results for the deuterated and 13C18O-labeled peptide with those from spectroscopic maps developed and used previously by the Skinner group. We find that the maps are typically accurate to within a few cm−1 for both frequencies and couplings, having larger errors only for the frequencies of terminal amides. PMID:24929378
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Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J
2014-03-15
Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Carr, J. K.; Roy, S.; Skinner, J. L.
2014-06-14
The spectroscopy of amide I vibrations has become a powerful tool for exploring protein structure and dynamics. To help with spectral interpretation, it is often useful to perform molecular dynamics (MD) simulations. To connect spectroscopic experiments to simulations in an efficient manner, several researchers have proposed “maps,” which relate observables in classical MD simulations to quantum spectroscopic variables. It can be difficult to discern whether errors in the theoretical results (compared to experiment) arise from inaccuracies in the MD trajectories or in the maps themselves. In this work, we evaluate spectroscopic maps independently from MD simulations by comparing experimental andmore » theoretical spectra for a single conformation of the α-helical model peptide Ac-Phe-(Ala){sub 5}-Lys-H{sup +} in the gas phase. Conformation-specific experimental spectra are obtained for the unlabeled peptide and for several singly and doubly {sup 13}C-labeled variants using infrared-ultraviolet double-resonance spectroscopy, and these spectra are found to be well-modeled by density functional theory (DFT) calculations at the B3LYP/6-31G** level. We then compare DFT results for the deuterated and {sup 13}C{sup 18}O-labeled peptide with those from spectroscopic maps developed and used previously by the Skinner group. We find that the maps are typically accurate to within a few cm{sup −1} for both frequencies and couplings, having larger errors only for the frequencies of terminal amides.« less
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Mapping the tumour human leukocyte antigen (HLA) ligandome by mass spectrometry.
Freudenmann, Lena Katharina; Marcu, Ana; Stevanović, Stefan
2018-07-01
The entirety of human leukocyte antigen (HLA)-presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide-specific immunotherapies, MS-based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution, and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and deconvolute multi-allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA-presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide-specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS-guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application, and provide an outlook on future developments. © 2018 John Wiley & Sons Ltd.
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ACTG: novel peptide mapping onto gene models.
Choi, Seunghyuk; Kim, Hyunwoo; Paek, Eunok
2017-04-15
In many proteogenomic applications, mapping peptide sequences onto genome sequences can be very useful, because it allows us to understand origins of the gene products. Existing software tools either take the genomic position of a peptide start site as an input or assume that the peptide sequence exactly matches the coding sequence of a given gene model. In case of novel peptides resulting from genomic variations, especially structural variations such as alternative splicing, these existing tools cannot be directly applied unless users supply information about the variant, either its genomic position or its transcription model. Mapping potentially novel peptides to genome sequences, while allowing certain genomic variations, requires introducing novel gene models when aligning peptide sequences to gene structures. We have developed a new tool called ACTG (Amino aCids To Genome), which maps peptides to genome, assuming all possible single exon skipping, junction variation allowing three edit distances from the original splice sites, exon extension and frame shift. In addition, it can also consider SNVs (single nucleotide variations) during mapping phase if a user provides the VCF (variant call format) file as an input. Available at http://prix.hanyang.ac.kr/ACTG/search.jsp . eunokpaek@hanyang.ac.kr. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
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van der Burgt, Yuri E M; Cobbaert, Christa M; Dalebout, Hans; Smit, Nico; Deelder, André M
2012-08-01
In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC-MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Copyright © 2012 Elsevier B.V. All rights reserved.
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High Throughput T Epitope Mapping and Vaccine Development
Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio
2010-01-01
Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Huang, Yingying; Triscari, Joseph M.; Tseng, George C.
Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides,more » a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s). Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and should be useful in algorithm development that employs improved models to predict fragment ion intensities.« less
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Kalyanaraman, Ananth; Cannon, William R; Latt, Benjamin; Baxter, Douglas J
2011-11-01
A MapReduce-based implementation called MR-MSPolygraph for parallelizing peptide identification from mass spectrometry data is presented. The underlying serial method, MSPolygraph, uses a novel hybrid approach to match an experimental spectrum against a combination of a protein sequence database and a spectral library. Our MapReduce implementation can run on any Hadoop cluster environment. Experimental results demonstrate that, relative to the serial version, MR-MSPolygraph reduces the time to solution from weeks to hours, for processing tens of thousands of experimental spectra. Speedup and other related performance studies are also reported on a 400-core Hadoop cluster using spectral datasets from environmental microbial communities as inputs. The source code along with user documentation are available on http://compbio.eecs.wsu.edu/MR-MSPolygraph. ananth@eecs.wsu.edu; william.cannon@pnnl.gov. Supplementary data are available at Bioinformatics online.
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NASA Astrophysics Data System (ADS)
Cai, Kaicong; Zheng, Xuan; Du, Fenfen
2017-08-01
The spectroscopy of amide-I vibrations has been widely utilized for the understanding of dynamical structure of polypeptides. For the modeling of amide-I spectra, two frequency maps were built for β-peptide analogue (N-ethylpropionamide, NEPA) in a number of solvents within different schemes (molecular mechanics force field based, GM map; DFT calculation based, GD map), respectively. The electrostatic potentials on the amide unit that originated from solvents and peptide backbone were correlated to the amide-I frequency shift from gas phase to solution phase during map parameterization. GM map is easier to construct with negligible computational cost since the frequency calculations for the samples are purely based on force field, while GD map utilizes sophisticated DFT calculations on the representative solute-solvent clusters and brings insight into the electronic structures of solvated NEPA and its chemical environments. The results show that the maps' predicted amide-I frequencies present solvation environmental sensitivities and exhibit their specific characters with respect to the map protocols, and the obtained vibrational parameters are in satisfactory agreement with experimental amide-I spectra of NEPA in solution phase. Although different theoretical schemes based maps have their advantages and disadvantages, the present maps show their potentials in interpreting the amide-I spectra for β-peptides, respectively.
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Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.
Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão
2018-05-24
The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.
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Spectra-first feature analysis in clinical proteomics - A case study in renal cancer.
Goh, Wilson Wen Bin; Wong, Limsoon
2016-10-01
In proteomics, useful signal may be unobserved or lost due to the lack of confident peptide-spectral matches. Selection of differential spectra, followed by associative peptide/protein mapping may be a complementary strategy for improving sensitivity and comprehensiveness of analysis (spectra-first paradigm). This approach is complementary to the standard approach where functional analysis is performed only on the finalized protein list assembled from identified peptides from the spectra (protein-first paradigm). Based on a case study of renal cancer, we introduce a simple spectra-binning approach, MZ-bin. We demonstrate that differential spectra feature selection using MZ-bin is class-discriminative and can trace relevant proteins via spectra associative mapping. Moreover, proteins identified in this manner are more biologically coherent than those selected directly from the finalized protein list. Analysis of constituent peptides per protein reveals high expression inconsistency, suggesting that the measured protein expressions are in fact, poor approximations of true protein levels. Moreover, analysis at the level of constituent peptides may provide higher resolution insight into the underlying biology: Via MZ-bin, we identified for the first time differential splice forms for the known renal cancer marker MAPT. We conclude that the spectra-first analysis paradigm is a complementary strategy to the traditional protein-first paradigm and can provide deeper level insight.
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Sachon, Emmanuelle; Nielsen, Per Franklin; Jensen, Ole Nørregaard
2007-06-01
Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.
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Rothan, Hussin A.; Bahrani, Hirbod; Mohamed, Zulqarnain; Abd Rahman, Noorsaadah; Yusof, Rohana
2014-01-01
Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities. PMID:24722532
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Free energy landscapes of a highly structured β-hairpin peptide and its single mutant
NASA Astrophysics Data System (ADS)
Kim, Eunae; Yang, Changwon; Jang, Soonmin; Pak, Youngshang
2008-10-01
We investigated the free energy landscapes of a highly structured β-hairpin peptide (MBH12) and a less structured peptide with a single mutation of Tyr6 to Asp6 (MBH10). For the free energy mapping, starting from an extended conformation, the replica exchange molecular dynamic simulations for two β-hairpins were performed using a modified version of an all-atom force field employing an implicit solvation (param99MOD5/GBSA). With the present simulation approach, we demonstrated that detailed stability changes associated with the sequence modification from MBH12 to MBH10 are quantitatively well predicted at the all-atom level.
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Cai, Kaicong; Zheng, Xuan; Du, Fenfen
2017-08-05
The spectroscopy of amide-I vibrations has been widely utilized for the understanding of dynamical structure of polypeptides. For the modeling of amide-I spectra, two frequency maps were built for β-peptide analogue (N-ethylpropionamide, NEPA) in a number of solvents within different schemes (molecular mechanics force field based, GM map; DFT calculation based, GD map), respectively. The electrostatic potentials on the amide unit that originated from solvents and peptide backbone were correlated to the amide-I frequency shift from gas phase to solution phase during map parameterization. GM map is easier to construct with negligible computational cost since the frequency calculations for the samples are purely based on force field, while GD map utilizes sophisticated DFT calculations on the representative solute-solvent clusters and brings insight into the electronic structures of solvated NEPA and its chemical environments. The results show that the maps' predicted amide-I frequencies present solvation environmental sensitivities and exhibit their specific characters with respect to the map protocols, and the obtained vibrational parameters are in satisfactory agreement with experimental amide-I spectra of NEPA in solution phase. Although different theoretical schemes based maps have their advantages and disadvantages, the present maps show their potentials in interpreting the amide-I spectra for β-peptides, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.
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Peptide adsorption on the hydrophobic surface: A free energy perspective
NASA Astrophysics Data System (ADS)
Sheng, Yuebiao; Wang, Wei; Chen, P.
2011-05-01
Protein adsorption is a very attractive topic which relates to many novel applications in biomaterials, biotechnology and nanotechnology. Ionic complementary peptides are a group of novel nano-biomaterials with many biomedical applications. In this work, molecular dynamics simulations of the ionic-complementary peptide EAK16-II on a hydrophobic graphite surface were performed under neutral, acidic and basic solution conditions. Adsorption free energy contour maps were obtained by analyzing the dynamical trajectories. Hydrophobic interactions were found to govern the adsorption of the first peptide molecule, and both hydrophobic and electrostatic interactions contributed to the adsorption of the second peptide molecule. Especially under acidic and basic solution conditions, interplay existed among chain-chain hydrophobic, chain-surface hydrophobic and chain-chain electrostatic interactions during the adsorption of the second peptide molecule. Non-charged residues were found to lie on the graphite surface, while charged residue side-chains oriented towards the solution after the peptide deposited on the surface. These results provide a basis for understanding peptide adsorption on the hydrophobic surface under different solution conditions, which is useful for novel applications such as bioactive implant devices and drug delivery material design.
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Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes.
McBride, Ryan; Head, Steven R; Ordoukhanian, Phillip; Law, Mansun
2016-01-01
With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface.
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Design and application of a data-independent precursor and product ion repository.
Thalassinos, Konstantinos; Vissers, Johannes P C; Tenzer, Stefan; Levin, Yishai; Thompson, J Will; Daniel, David; Mann, Darrin; DeLong, Mark R; Moseley, M Arthur; America, Antoine H; Ottens, Andrew K; Cavey, Greg S; Efstathiou, Georgios; Scrivens, James H; Langridge, James I; Geromanos, Scott J
2012-10-01
The functional design and application of a data-independent LC-MS precursor and product ion repository for protein identification, quantification, and validation is conceptually described. The ion repository was constructed from the sequence search results of a broad range of discovery experiments investigating various tissue types of two closely related mammalian species. The relative high degree of similarity in protein complement, ion detection, and peptide and protein identification allows for the analysis of normalized precursor and product ion intensity values, as well as standardized retention times, creating a multidimensional/orthogonal queryable, qualitative, and quantitative space. Peptide ion map selection for identification and quantification is primarily based on replication and limited variation. The information is stored in a relational database and is used to create peptide- and protein-specific fragment ion maps that can be queried in a targeted fashion against the raw or time aligned ion detections. These queries can be conducted either individually or as groups, where the latter affords pathway and molecular machinery analysis of the protein complement. The presented results also suggest that peptide ionization and fragmentation efficiencies are highly conserved between experiments and practically independent of the analyzed biological sample when using similar instrumentation. Moreover, the data illustrate only minor variation in ionization efficiency with amino acid sequence substitutions occurring between species. Finally, the data and the presented results illustrate how LC-MS performance metrics can be extracted and utilized to ensure optimal performance of the employed analytical workflows.
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Tan, Yaw Sing; Spring, David R; Abell, Chris; Verma, Chandra S
2015-07-14
A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein-protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules.
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Ko, Yoo-Jin; Kwon, Kil-Young; Kum, Kee-Yeon; Lee, Woo-Cheol; Baek, Seung-Ho; Kang, Mo K.; Shon, Won-Jun
2015-01-01
Porphyromonas gingivalis is considered with inducing pulpal inflammation and has lipopolysaccharide (LPS) as an inflammatory stimulator. GV1001 peptide has anticancer and anti-inflammation activity due to inhibiting activation of signaling molecules after penetration into the various types of cells. Therefore, this study examined inhibitory effect of GV1001 on dental pulp cells (hDPCs) stimulated by P. gingivalis LPS. The intracellular distribution of GV1001 was analyzed by confocal microscopy. Real-time RT-PCR was performed to determine the expression levels of TNF-α and IL-6 cytokines. The role of signaling by MAP kinases (ERK and p38) was explored using Western blot analysis. The effect of GV1001 peptide on hDPCs viability was measured by MTT assay. GV1001 was predominantly located in hDPC cytoplasm. The peptide inhibited P. gingivalis LPS-induced TNF-α and IL-6 production in hDPCs without significant cytotoxicity. Furthermore, GV1001 treatment markedly inhibited the phosphorylation of MAP kinases (ERK and p38) in LPS-stimulated hDPCs. GV1001 may prevent P. gingivalis LPS-induced inflammation of apical tissue. Also, these findings provide mechanistic insight into how GV1001 peptide causes anti-inflammatory actions in LPS-stimulated pulpitis without significantly affecting cell viability. PMID:26604431
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Kim, Tae-Hyung; El-Said, Waleed Ahmed; An, Jeung Hee; Choi, Jeong-Woo
2013-04-01
A cell chip composed of ITO, gold nanoparticles (GNP) and RGD-MAP-C peptide composites was fabricated to enhance the electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The structural characteristics of the fabricated surfaces were confirmed by both scanning electron microscopy and surface-enhanced Raman spectroscopy. HB1.F3 cells were allowed to attach to various composites electrodes in the cell chip and the material-dependent effects on electrochemical signals and cell proliferation were analyzed. The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to be the best material in regards to enhancing the voltammetric signals of HB1.F3 cells when exposed to cyclic voltammetry, as well as for increasing cell proliferation. Differential pulse voltammetry was performed to evaluate the adverse effects of doxorubicin on HB1.F3 cells. In these experiments, negative correlations between cell viability and chemical concentrations were obseved, which were more sensitive than MTT viability assay especially at low concentrations (<0.1 μg/mL). In this basic science study, a cell chip composed of ITO, gold nanoparticles and RGD-MAP-C peptide composites was fabricated to enhance electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to best enhance the voltammetric signals of the studied cells. Copyright © 2013 Elsevier Inc. All rights reserved.
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Design and synthesis of multiple antigenic peptides and their application for dengue diagnosis.
Rai, Reeta; Dubey, Sameer; Santosh, K V; Biswas, Ashutosh; Mehrotra, Vinit; Rao, D N
2017-09-01
Major difficulty in development of dengue diagnostics is availability of suitable antigens. To overcome this, we made an attempt to develop a peptide based diagnosis which offers significant advantage over other methods. With the help of in silico methods, two epitopes were selected from envelope protein and three from NS1 protein of dengue virus. These were synthesized in combination as three multiple antigenic peptides (MAPs). We have tested 157 dengue positive sera confirmed for NS1 antigen. MAP1 showed 96.81% sera positive for IgM and 68.15% positive for IgG. MAP2 detected 94.90% IgM and 59.23% IgG positive sera. MAP3 also detected 96.17% IgM and 59.87% IgG positive sera. To the best of our knowledge this is the first study describing the use of synthetic multiple antigenic peptides for the diagnosis of dengue infection. This study describes MAPs as a promising tool for the use in serodiagnosis of dengue. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
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Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán
2015-01-01
Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409
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Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán
2015-07-01
Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Analysis of protein glycation products by MALDI-TOF/MS.
Kislinger, Thomas; Humeny, Andreas; Peich, Carlo C; Becker, Cord-Michael; Pischetsrieder, Monika
2005-06-01
Matrix-assisted laser desorption ionization-mass spectrometry with time-of-flight detection (MALDI-TOF/MS) is a promising tool to analyze advanced glycation end product (AGE)-modified proteins. The combination of soft ionization (MALDI) with time-of-flight mass detection allows analysis of peptides and proteins of a molecular mass up to 300 kDa with minimal sample workup. Because the direct structural analysis of intact AGE proteins is not possible due to the formation of broad and poorly resolved peaks, peptide mapping was introduced into the analysis of AGE proteins by MALDI-TOF/MS, allowing site-specific analysis of defined AGEs. When methylglyoxal-modified lysozyme was subjected to MALDI-TOF/MS peptide mapping, methylimidazolone and argpyrimidine attached to the arginine residue and carboxyethyl (CEL) bound to the lysine were detected on peptide(aa1-7) (KVFGRCE). In contrast, only one methylimidazolone was found on peptide(aa8-35) (LAAAMKRHGLDNYRGYSLGNWVCAAKFE) and peptide(aa120-129) (VQAWIRGCRL), respectively. The analysis of AGE protein, which had been incubated with glucose, revealed the presence of an Amadori product and a carboxymethyl residue (CML) on peptide(aa1-7) and peptide(aa8-35), as well as an imidazolone A on peptide(aa120-129). Furthermore, the early Maillard reaction of lysozyme, which had been glycated by seven different sugars, was monitored by MALDI-TOF/MS peptide mapping. Finally, this approach was successfully applied for site- and product-specific relative quantification of AGEs. For example, kinetics of CML and Amadori product formation on peptide(aa1-7), as well as imidazolone A formation on peptide(aa120-129), were determined.
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Xia, Yitian; Shang, Yuan; Zhang, Rongguang; Zhu, Jinwei
2017-08-10
The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
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Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V
2013-11-19
As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.
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Mameli, G; Madeddu, G; Cossu, D; Galleri, G; Manetti, R; Babudieri, S; Mura, M Stella; Sechi, L A
2016-01-01
Infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) has been associated with increased risk of multiple sclerosis (MS). However, the mechanism linking these pathologies is unclear. Different reports indicate the association of EBV, and recently Mycobacterium avium subsp. paratuberculosis (MAP), with MS. For a better understanding of the role of these pathogens, the host response induced by selected antigenic peptides in subjects with a history of IM that significantly increases the risk of MS was investigated. Both humoral and cell-mediated response against peptides able to induce a specific immune activation in MS patients deriving from lytic and latent EBV antigens BOLF1(305-320), EBNA1(400-413), from MAP MAP_4027(18-32), MAP_0106c(121-132) and from human proteins IRF5(424-434) and MBP(85-98) in subjects with current and past IM were examined. EBNA1 and MAP_0106c peptides were able to induce a humoral immune response in subjects with a history of clinical IM in an independent manner. Moreover, these peptides were capable of inducing pro-inflammatory cytokine interferon γ by CD4+ and CD8+ T lymphocytes and interleukin 6 and tumour necrosis factor α by CD14+ monocyte cells. Our results highlight that EBV and MAP may be involved independently in the same causal process leading to MS in subjects with a history of IM. © 2015 EAN.
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Plant peptides in defense and signaling.
Marmiroli, Nelson; Maestri, Elena
2014-06-01
This review focuses on plant peptides involved in defense against pathogen infection and those involved in the regulation of growth and development. Defense peptides, defensins, cyclotides and anti-microbial peptides are compared and contrasted. Signaling peptides are classified according to their major sites of activity. Finally, a network approach to creating an interactomic peptide map is described. Copyright © 2014 Elsevier Inc. All rights reserved.
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Translocation of cell-penetrating peptides into Candida fungal pathogens.
Gong, Zifan; Karlsson, Amy J
2017-09-01
Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF) 3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF) 3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens. © 2017 The Protein Society.
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Chakraborty, Asish B.; Berger, Scott J.
2005-01-01
The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described. PMID:16522853
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Curciarello, Renata; Smaldini, Paola L; Candreva, Angela M; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A; Petruccelli, Silvana; Docena, Guillermo H
2014-01-01
Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.
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The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.
Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun
2016-11-01
Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
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Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus
2013-01-01
We have recently developed a high-density photolithographic, peptide array technology with a theoretical upper limit of 2 million different peptides per array of 2 cm(2). Here, we have used this to perform complete and exhaustive analyses of linear B cell epitopes of a medium sized protein target using human serum albumin (HSA) as an example. All possible overlapping 15-mers from HSA were synthesized and probed with a commercially available polyclonal rabbit anti-HSA antibody preparation. To allow for identification of even the weakest epitopes and at the same time perform a detailed characterization of key residues involved in antibody binding, the array also included complete single substitution scans (i.e. including each of the 20 common amino acids) at each position of each 15-mer peptide. As specificity controls, all possible 15-mer peptides from bovine serum albumin (BSA) and from rabbit serum albumin (RSA) were included as well. The resulting layout contained more than 200.000 peptide fields and could be synthesized in a single array on a microscope slide. More than 20 linear epitope candidates were identified and characterized at high resolution i.e. identifying which amino acids in which positions were needed, or not needed, for antibody interaction. As expected, moderate cross-reaction with some peptides in BSA was identified whereas no cross-reaction was observed with peptides from RSA. We conclude that high-density peptide microarrays are a very powerful methodology to identify and characterize linear antibody epitopes, and should advance detailed description of individual specificities at the single antibody level as well as serologic analysis at the proteome-wide level.
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Dinosaur peptides suggest mechanisms of protein survival.
San Antonio, James D; Schweitzer, Mary H; Jensen, Shane T; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P R O
2011-01-01
Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.
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Complexity of free energy landscapes of peptides revealed by nonlinear principal component analysis.
Nguyen, Phuong H
2006-12-01
Employing the recently developed hierarchical nonlinear principal component analysis (NLPCA) method of Saegusa et al. (Neurocomputing 2004;61:57-70 and IEICE Trans Inf Syst 2005;E88-D:2242-2248), the complexities of the free energy landscapes of several peptides, including triglycine, hexaalanine, and the C-terminal beta-hairpin of protein G, were studied. First, the performance of this NLPCA method was compared with the standard linear principal component analysis (PCA). In particular, we compared two methods according to (1) the ability of the dimensionality reduction and (2) the efficient representation of peptide conformations in low-dimensional spaces spanned by the first few principal components. The study revealed that NLPCA reduces the dimensionality of the considered systems much better, than did PCA. For example, in order to get the similar error, which is due to representation of the original data of beta-hairpin in low dimensional space, one needs 4 and 21 principal components of NLPCA and PCA, respectively. Second, by representing the free energy landscapes of the considered systems as a function of the first two principal components obtained from PCA, we obtained the relatively well-structured free energy landscapes. In contrast, the free energy landscapes of NLPCA are much more complicated, exhibiting many states which are hidden in the PCA maps, especially in the unfolded regions. Furthermore, the study also showed that many states in the PCA maps are mixed up by several peptide conformations, while those of the NLPCA maps are more pure. This finding suggests that the NLPCA should be used to capture the essential features of the systems. (c) 2006 Wiley-Liss, Inc.
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Predicting PDZ domain mediated protein interactions from structure
2013-01-01
Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on training–testing domain sequence similarity. Using both predictors, we defined a functional map of human PDZ domain biology and predict novel PDZ domain function. Users may access our structure-based and previous sequence-based predictors at http://webservice.baderlab.org/domains/POW. PMID:23336252
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Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry
NASA Astrophysics Data System (ADS)
Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad
2015-12-01
A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.
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Souza, Cleverson D; Bannantine, John P; Brown, Wendy C; Norton, M Grant; Davis, William C; Hwang, Julianne K; Ziaei, Parissa; Abdellrazeq, Gaber S; Eren, Meaghan V; Deringer, James R; Laws, Elizabeth; Cardieri, Maria Clara D
2017-05-14
We evaluated the potential of a nanoparticle (NP) delivery system to improve methods of delivery of candidate peptide-based vaccines for Paratuberculosis in cattle. Peptides derived from Mycobacterium avium subsp. paratuberculosis (Map), and the pro-inflammatory monophosphoryl lipid A (MPLA) were incorporated in polymeric NPs based on poly (d,l-lactide-co-glycolide) (PLGA). The PLGA/MPLA NPs carriers were incubated with macrophages to examine their effects on survival and function. PLGA/MPLA NPs, with and without Map antigens, are efficiently phagocytized by macrophages with no evidence of toxicity. PLGA/MPLA NP formulations did not alter the level of expression of MHC I or II molecules. Expression of TNFα and IL12p40 was increased in Map-loaded NPs. T-cell proliferation studies using a model peptide from Anaplasma marginale demonstrated that a CD4 T-cell recall response could be elicited with macrophages pulsed with the peptide encapsulated in the PLGA/MPLA NP. These findings indicate PLGA/MPLA NPs can be used as a vehicle for delivery and testing of candidate peptide-based vaccines. These results will assist on more in depth studies on PLGA NP delivery systems that may lead to the development of a peptide-based vaccine for cattle. © 2017 The Society for Applied Microbiology.
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Migliolo, Ludovico; Silva, Osmar N.; Silva, Paula A.; Costa, Maysa P.; Costa, Carolina R.; Nolasco, Diego O.; Barbosa, João A. R. G.; Silva, Maria R. R.; Bemquerer, Marcelo P.; Lima, Lidia M. P.; Romanos, Maria T. V.; Freitas, Sonia M.; Magalhães, Beatriz S.; Franco, Octavio L.
2012-01-01
Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets. PMID:23056574
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Aiyetan, Paul; Zhang, Bai; Zhang, Zhen; Zhang, Hui
2014-01-01
Mass spectrometry based glycoproteomics has become a major means of identifying and characterizing previously N-linked glycan attached loci (glycosites). In the bottom-up approach, several factors which include but not limited to sample preparation, mass spectrometry analyses, and protein sequence database searches result in previously N-linked peptide spectrum matches (PSMs) of varying lengths. Given that multiple PSM scan map to a glycosite, we reason that identified PSMs are varying length peptide species of a unique set of glycosites. Because associated spectra of these PSMs are typically summed separately, true glycosite associated spectra counts are lost or complicated. Also, these varying length peptide species complicate protein inference as smaller sized peptide sequences are more likely to map to more proteins than larger sized peptides or actual glycosite sequences. Here, we present XGlycScan. XGlycScan maps varying length peptide species to glycosites to facilitate an accurate quantification of glycosite associated spectra counts. We observed that this reduced the variability in reported identifications of mass spectrometry technical replicates of our sample dataset. We also observed that mapping identified peptides to glycosites provided an assessment of search-engine identification. Inherently, XGlycScan reported glycosites reduce the complexity in protein inference. We implemented XGlycScan in the platform independent Java programing language and have made it available as open source. XGlycScan's source code is freely available at https://bitbucket.org/paiyetan/xglycscan/src and its compiled binaries and documentation can be freely downloaded at https://bitbucket.org/paiyetan/xglycscan/downloads. The graphical user interface version can also be found at https://bitbucket.org/paiyetan/xglycscangui/src and https://bitbucket.org/paiyetan/xglycscangui/downloads respectively.
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Hunt, Katharine F; Dunn, Joel T; le Roux, Carel W; Reed, Laurence J; Marsden, Paul K; Patel, Ameet G; Amiel, Stephanie A
2016-10-01
Improved appetite control, possibly mediated by exaggerated gut peptide responses to eating, may contribute to weight loss after Roux-en-Y gastric bypass (RYGB). This study compared brain responses to food ingestion between post-RYGB (RYGB), normal weight (NW), and obese (Ob) unoperated subjects and explored the role of gut peptide responses in RYGB. Neuroimaging with [(18)F]-fluorodeoxyglucose (FDG) positron emission tomography was performed in 12 NW, 21 Ob, and 9 RYGB (18 ± 13 months postsurgery) subjects after an overnight fast, once FED (400 kcal mixed meal), and once FASTED, in random order. RYGB subjects repeated the studies with somatostatin infusion and basal insulin replacement. Fullness, sickness, and postscan ad libitum meal consumption were measured. Regional brain FDG uptake was compared using statistical parametric mapping. RYGB subjects had higher overall fullness and food-induced sickness and lower ad libitum consumption. Brain responses to eating differed in the hypothalamus and pituitary (exaggerated activation in RYGB), left medial orbital cortex (OC) (activation in RYGB, deactivation in NW), right dorsolateral frontal cortex (deactivation in RYGB and NW, absent in Ob), and regions mapping to the default mode network (exaggerated deactivation in RYGB). Somatostatin in RYGB reduced postprandial gut peptide responses, sickness, and medial OC activation. RYGB induces weight loss by augmenting normal brain responses to eating in energy balance regions, restoring lost inhibitory control, and altering hedonic responses. Altered postprandial gut peptide responses primarily mediate changes in food-induced sickness and OC responses, likely to associate with food avoidance. © 2016 by the American Diabetes Association.
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Picotti, Paola; Clement-Ziza, Mathieu; Lam, Henry; Campbell, David S.; Schmidt, Alexander; Deutsch, Eric W.; Röst, Hannes; Sun, Zhi; Rinner, Oliver; Reiter, Lukas; Shen, Qin; Michaelson, Jacob J.; Frei, Andreas; Alberti, Simon; Kusebauch, Ulrike; Wollscheid, Bernd; Moritz, Robert; Beyer, Andreas; Aebersold, Ruedi
2013-01-01
Complete reference maps or datasets, like the genomic map of an organism, are highly beneficial tools for biological and biomedical research. Attempts to generate such reference datasets for a proteome so far failed to reach complete proteome coverage, with saturation apparent at approximately two thirds of the proteomes tested, even for the most thoroughly characterized proteomes. Here, we used a strategy based on high-throughput peptide synthesis and mass spectrometry to generate a close to complete reference map (97% of the genome-predicted proteins) of the S. cerevisiae proteome. We generated two versions of this mass spectrometric map one supporting discovery- (shotgun) and the other hypothesis-driven (targeted) proteomic measurements. The two versions of the map, therefore, constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. The reference libraries can be browsed via a web-based repository and associated navigation tools. To demonstrate the utility of the reference libraries we applied them to a protein quantitative trait locus (pQTL) analysis, which requires measurement of the same peptides over a large number of samples with high precision. Protein measurements over a set of 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, impacting on the levels of related proteins. Our results suggest that selective pressure favors the acquisition of sets of polymorphisms that maintain the stoichiometry of protein complexes and pathways. PMID:23334424
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Imholte, Gregory; Gottardo, Raphael
2017-01-01
Summary The peptide microarray immunoassay simultaneously screens sample serum against thousands of peptides, determining the presence of antibodies bound to array probes. Peptide microarrays tiling immunogenic regions of pathogens (e.g. envelope proteins of a virus) are an important high throughput tool for querying and mapping antibody binding. Because of the assay’s many steps, from probe synthesis to incubation, peptide microarray data can be noisy with extreme outliers. In addition, subjects may produce different antibody profiles in response to an identical vaccine stimulus or infection, due to variability among subjects’ immune systems. We present a robust Bayesian hierarchical model for peptide microarray experiments, pepBayes, to estimate the probability of antibody response for each subject/peptide combination. Heavy-tailed error distributions accommodate outliers and extreme responses, and tailored random effect terms automatically incorporate technical effects prevalent in the assay. We apply our model to two vaccine trial datasets to demonstrate model performance. Our approach enjoys high sensitivity and specificity when detecting vaccine induced antibody responses. A simulation study shows an adaptive thresholding classification method has appropriate false discovery rate control with high sensitivity, and receiver operating characteristics generated on vaccine trial data suggest that pepBayes clearly separates responses from non-responses. PMID:27061097
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Can single molecule localization microscopy be used to map closely spaced RGD nanodomains?
Nicovich, Philip R.; Soeriyadi, Alexander; Nieves, Daniel J.; Gooding, J. Justin; Gaus, Katharina
2017-01-01
Cells sense and respond to nanoscale variations in the distribution of ligands to adhesion receptors. This makes single molecule localization microscopy (SMLM) an attractive tool to map the distribution of ligands on nanopatterned surfaces. We explore the use of SMLM spatial cluster analysis to detect nanodomains of the cell adhesion-stimulating tripeptide arginine-glycine-aspartic acid (RGD). These domains were formed by the phase separation of block copolymers with controllable spacing on the scale of tens of nanometers. We first determined the topology of the block copolymer with atomic force microscopy (AFM) and then imaged the localization of individual RGD peptides with direct stochastic optical reconstruction microscopy (dSTORM). To compare the data, we analyzed the dSTORM data with DBSCAN (density-based spatial clustering application with noise). The ligand distribution and polymer topology are not necessary identical since peptides may attach to the polymer outside the nanodomains and/or coupling and detection of peptides within the nanodomains is incomplete. We therefore performed simulations to explore the extent to which nanodomains could be mapped with dSTORM. We found that successful detection of nanodomains by dSTORM was influenced by the inter-domain spacing and the localization precision of individual fluorophores, and less by non-specific absorption of ligands to the substratum. For example, under our imaging conditions, DBSCAN identification of nanodomains spaced further than 50 nm apart was largely independent of background localisations, while nanodomains spaced closer than 50 nm required a localization precision of ~11 nm to correctly estimate the modal nearest neighbor distance (NDD) between nanodomains. We therefore conclude that SMLM is a promising technique to directly map the distribution and nanoscale organization of ligands and would benefit from an improved localization precision. PMID:28723958
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Samgina, Tatyana Yu; Gorshkov, Vladimir A; Artemenko, Konstantin A; Vorontsov, Egor A; Klykov, Oleg V; Ogourtsov, Sergey V; Zubarev, Roman A; Lebedev, Albert T
2012-04-01
Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC-MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required. Copyright © 2012 Elsevier Inc. All rights reserved.
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Dinosaur Peptides Suggest Mechanisms of Protein Survival
San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P. R. O.
2011-01-01
Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a ‘preservation motif’, and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival. PMID:21687667
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Dinosaur Peptides Suggest Mechanisms of Protein Survival
DOE Office of Scientific and Technical Information (OSTI.GOV)
San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.
Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results showmore » empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.« less
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Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B; Holo, Helge; Nes, Ingolf F
2010-11-01
The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota.
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Curciarello, Renata; Smaldini, Paola L.; Candreva, Angela M.; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A.
2014-01-01
Background Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Methods Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Results Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Conclusions Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide. PMID:24416141
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Sintsova, Oksana; Gladkikh, Irina; Chausova, Victoria; Monastyrnaya, Margarita; Anastyuk, Stanislav; Chernikov, Oleg; Yurchenko, Ekaterina; Aminin, Dmitriy; Isaeva, Marina; Leychenko, Elena; Kozlovskaya, Emma
2018-02-20
Sea anemone mucus, due to its multiple and vital functions, is a valuable substance for investigation of new biologically active peptides. In this work, compounds of Heteractis magnifica mucus were separated by multistage liquid chromatography and resulting fractions were analyzed by MALDI-TOF MS. Peptide maps constructed according to the molecular masses and hydrophobicity showed presence of 326 both new and known peptides. Several major peptides from mucus were identified, including the sodium channel toxin RpII isolated earlier from H. magnifica, and four Kunitz-type proteinase inhibitors identical to H. crispa ones. Kunitz-type transcript diversity was studied and sequences of mature peptides were deduced. New β-defensin α-amylase inhibitor, a homolog of helianthamide from Stichodactyla helianthus, was isolated and structurally characterized. Overall, H. magnifica is a source of biologically active peptides with great pharmacological potential. Proteinase and α-amylase inhibitors along with toxins are major components of H. magnifica mucus which play an important role in the successful existence of sea anemones. Obtained peptide maps create a basis for more accurate identification of peptides during future transcriptomic/genomic studies of sea anemone H. magnifica. Copyright © 2017 Elsevier B.V. All rights reserved.
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Koromyslova, Anna D; Chugunov, Anton O; Efremov, Roman G
2014-04-28
Molecular surfaces are the key players in biomolecular recognition and interactions. Nowadays, it is trivial to visualize a molecular surface and surface-distributed properties in three-dimensional space. However, such a representation trends to be biased and ambiguous in case of thorough analysis. We present a new method to create 2D spherical projection maps of entire protein surfaces and manipulate with them--protein surface topography (PST). It permits visualization and thoughtful analysis of surface properties. PST helps to easily portray conformational transitions, analyze proteins' properties and their dynamic behavior, improve docking performance, and reveal common patterns and dissimilarities in molecular surfaces of related bioactive peptides. This paper describes basic usage of PST with an example of small G-proteins conformational transitions, mapping of caspase-1 intersubunit interface, and intrinsic "complementarity" in the conotoxin-acetylcholine binding protein complex. We suggest that PST is a beneficial approach for structure-function studies of bioactive peptides and small proteins.
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NASA Astrophysics Data System (ADS)
van Oosten, Luuk N.; Pieterse, Mervin; Pinkse, Martijn W. H.; Verhaert, Peter D. E. M.
2015-12-01
Animal venoms and toxins are a valuable source of bioactive peptides with pharmacologic relevance as potential drug leads. A large subset of biologically active peptides discovered up till now contain disulfide bridges that enhance stability and activity. To discover new members of this class of peptides, we developed a workflow screening specifically for those peptides that contain inter- and intra-molecular disulfide bonds by means of three-dimensional (3D) mass mapping. Two intrinsic properties of the sulfur atom, (1) its relatively large negative mass defect, and (2) its isotopic composition, allow for differentiation between cysteine-containing peptides and peptides lacking sulfur. High sulfur content in a peptide decreases the normalized nominal mass defect (NMD) and increases the normalized isotopic shift (NIS). Hence in a 3D plot of mass, NIS, and NMD, peptides with sulfur appear in this plot with a distinct spatial localization compared with peptides that lack sulfur. In this study we investigated the skin secretion of two frog species; Odorrana schmackeri and Bombina variegata. Peptides from the crude skin secretions were separated by nanoflow LC, and of all eluting peptides high resolution zoom scans were acquired in order to accurately determine both monoisotopic mass and average mass. Both the NMD and the NIS were calculated from the experimental data using an in-house developed MATLAB script. Candidate peptides exhibiting a low NMD and high NIS values were selected for targeted de novo sequencing, and this resulted in the identification of several novel inter- and intra-molecular disulfide bond containing peptides.
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A polyalanine peptide derived from polar fish with anti-infectious activities
NASA Astrophysics Data System (ADS)
Cardoso, Marlon H.; Ribeiro, Suzana M.; Nolasco, Diego O.; de La Fuente-Núñez, César; Felício, Mário R.; Gonçalves, Sónia; Matos, Carolina O.; Liao, Luciano M.; Santos, Nuno C.; Hancock, Robert E. W.; Franco, Octávio L.; Migliolo, Ludovico
2016-02-01
Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.
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Chemokine interactome mapping enables tailored intervention in acute and chronic inflammation.
von Hundelshausen, Philipp; Agten, Stijn M; Eckardt, Veit; Blanchet, Xavier; Schmitt, Martin M; Ippel, Hans; Neideck, Carlos; Bidzhekov, Kiril; Leberzammer, Julian; Wichapong, Kanin; Faussner, Alexander; Drechsler, Maik; Grommes, Jochen; van Geffen, Johanna P; Li, He; Ortega-Gomez, Almudena; Megens, Remco T A; Naumann, Ronald; Dijkgraaf, Ingrid; Nicolaes, Gerry A F; Döring, Yvonne; Soehnlein, Oliver; Lutgens, Esther; Heemskerk, Johan W M; Koenen, Rory R; Mayo, Kevin H; Hackeng, Tilman M; Weber, Christian
2017-04-05
Chemokines orchestrate leukocyte trafficking and function in health and disease. Heterophilic interactions between chemokines in a given microenvironment may amplify, inhibit, or modulate their activity; however, a systematic evaluation of the chemokine interactome has not been performed. We used immunoligand blotting and surface plasmon resonance to obtain a comprehensive map of chemokine-chemokine interactions and to confirm their specificity. Structure-function analyses revealed that chemokine activity can be enhanced by CC-type heterodimers but inhibited by CXC-type heterodimers. Functional synergism was achieved through receptor heteromerization induced by CCL5-CCL17 or receptor retention at the cell surface via auxiliary proteoglycan binding of CCL5-CXCL4. In contrast, inhibitory activity relied on conformational changes (in CXCL12), affecting receptor signaling. Obligate CC-type heterodimers showed high efficacy and potency and drove acute lung injury and atherosclerosis, processes abrogated by specific CCL5-derived peptide inhibitors or knock-in of an interaction-deficient CXCL4 variant. Atheroprotective effects of CCL17 deficiency were phenocopied by a CCL5-derived peptide disrupting CCL5-CCL17 heterodimers, whereas a CCL5 α-helix peptide mimicked inhibitory effects on CXCL12-driven platelet aggregation. Thus, formation of specific chemokine heterodimers differentially dictates functional activity and can be exploited for therapeutic targeting. Copyright © 2017, American Association for the Advancement of Science.
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Subramanian, Sarada; Andal, S; Karande, Anjali A; Radhakantha Adiga, P
2003-11-07
This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.
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Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*
Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard
2016-01-01
X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949
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An open-source computational and data resource to analyze digital maps of immunopeptidomes
Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J.; ...
2015-07-08
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.
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Bøhle, Liv Anette; Brede, Dag Anders; Diep, Dzung B.; Holo, Helge; Nes, Ingolf F.
2010-01-01
The intestinal flora of mammals contains lactic acid bacteria (LAB) that may provide positive health effects for the host. Such bacteria are referred to as probiotic bacteria. From a pig, we have isolated a Lactobacillus reuteri strain that produces an antimicrobial peptide (AMP). The peptide was purified and characterized, and it was unequivocally shown that the AMP was a well-defined degradation product obtained from the mucus adhesion-promoting protein (MapA); it was therefore termed AP48-MapA. This finding demonstrates how large proteins might inherit unexpected pleiotropic functions by conferring antimicrobial capacities on the producer. The MapA/AP48-MapA system is the first example where a large protein of an intestinal LAB is shown to give rise to such an AMP. It is also of particular interest that the protein that provides this AMP is associated with the binding of the bacterium producing it to the surface/lining of the gut. This finding gives us new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota. PMID:20833791
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Selective detection of thiosulfate-containing peptides using tandem mass spectrometry.
Raftery, Mark J
2005-01-01
Incubation of proteins or peptides containing disulfide bonds (S-S) with sodium sulfite (Na(2)SO(3)) cleaves S-S bonds producing approximately equimolar amounts of free thiols (-SH) and thiosulfates (-S-SO(3)H), a process known as sulfitolysis. Proteins and peptides containing thiosulfates were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry (MS) and peptide mapping. The mass of the thiosulfate-containing peptide formed from oxidized insulin B chain was 3478.02 Da, 80 Da greater than the reduced peptide and corresponding precisely to addition of sulfur trioxide (SO(3)). Disulfide bond cleavage was also observed using RP-HPLC and MS after incubation of the intramolecular homodimer of mouse S100A8 (mass 20614 Da). The mass of HPLC-separated A8-SH was 10308 Da, and 10388 Da for A8-S-SO(3)H. Loss of SO(3) from multiply charged precursor ions was generally observed at elevated declustering potentials in the source region or within q(2) at relatively low collision energies (approximately 20 V). The characteristic loss of SO(3) at low collision energies preceded peptide backbone fragmentations at higher collision energies. Accurate mass measurement and charge-state discrimination, using a hybrid quadrupole time-of-flight mass spectrometer, allowed specific detection of thiosulfate-containing peptides. An information-dependent acquisition method, where the switch criterion was loss of m/z 79.9568, specifically identified 11 thiosulfate-containing peptides using nano-LC/MS from a tryptic digest of bovine serum albumin (BSA).
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Structure and dynamics of the peptide strand KRFK from the thrombospondin TSP-1 in water.
Taleb Bendiab, W; Benomrane, B; Bounaceur, B; Dauchez, M; Krallafa, A M
2018-02-14
Theoretical investigations of a solute in liquid water at normal temperature and pressure can be performed at different levels of theory. Static quantum calculations as well as classical and ab initio molecular dynamics are used to completely explore the conformational space for large solvated molecular systems. In the classical approach, it is essential to describe all of the interactions of the solute and the solvent in detail. Water molecules are very often described as rigid bodies when the most commonly used interaction potentials, such as the SPCE and the TIP4P models, are employed. Recently, a physical model based upon a cluster of rigid water molecules with a tetrahedral architecture (AB 4 ) was proposed that describes liquid water as a mixture of both TIP4P and SPCE molecular species that occur in the proportions implied by the tetrahedral architecture (one central molecule versus four outer molecules; i.e., 20% TIP4P versus 80% SPCE molecules). In this work, theoretical spectroscopic data for a peptide strand were correlated with the structural properties of the peptide strand solvated in water, based on data calculated using different theoretical approaches and physical models. We focused on a particular peptide strand, KRFK (lysine-arginine-phenylalanine-lysine), found in the thrombospondin TSP-1, due to its interesting properties. As the activity and electronic structure of this system is strongly linked to its structure, we correlated its structure with charge-density maps obtained using different semi-empirical charge Q eq equations. The structural and thermodynamic properties obtained from classical simulations were correlated with ab initio molecular dynamics (AIMD) data. Structural changes in the peptide strand were rationalized in terms of the motions of atoms and groups of atoms. To achieve this, conformational changes were investigated using calculated infrared spectra for the peptide in the gas phase and in water solvent. The calculated AIMD infrared spectrum for the peptide was correlated with static quantum calculations of the molecular system based on a harmonic approach as well as the VDOS (vibrational density of states) spectra obtained using various classical solvent models (SPCE, TIP4P, and AB 4 ) and charge maps.
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Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C
1997-06-01
The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.
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Piloting the membranolytic activities of peptides with a self-organizing map.
Lin, Yen-Chu; Hiss, Jan A; Schneider, Petra; Thelesklaf, Peter; Lim, Yi Fan; Pillong, Max; Koehler, Fabian M; Dittrich, Petra S; Halin, Cornelia; Wessler, Silja; Schneider, Gisbert
2014-10-13
Antimicrobial peptides (AMPs) show remarkable selectivity toward lipid membranes and possess promising antibiotic potential. Their modes of action are diverse and not fully understood, and innovative peptide design strategies are needed to generate AMPs with improved properties. We present a de novo peptide design approach that resulted in new AMPs possessing low-nanomolar membranolytic activities. Thermal analysis revealed an entropy-driven mechanism of action. The study demonstrates sustained potential of advanced computational methods for designing peptides with the desired activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Designing Antibacterial Peptides with Enhanced Killing Kinetics
Waghu, Faiza H.; Joseph, Shaini; Ghawali, Sanket; Martis, Elvis A.; Madan, Taruna; Venkatesh, Kareenhalli V.; Idicula-Thomas, Susan
2018-01-01
Antimicrobial peptides (AMPs) are gaining attention as substitutes for antibiotics in order to combat the risk posed by multi-drug resistant pathogens. Several research groups are engaged in design of potent anti-infective agents using natural AMPs as templates. In this study, a library of peptides with high sequence similarity to Myeloid Antimicrobial Peptide (MAP) family were screened using popular online prediction algorithms. These peptide variants were designed in a manner to retain the conserved residues within the MAP family. The prediction algorithms were found to effectively classify peptides based on their antimicrobial nature. In order to improve the activity of the identified peptides, molecular dynamics (MD) simulations, using bilayer and micellar systems could be used to design and predict effect of residue substitution on membranes of microbial and mammalian cells. The inference from MD simulation studies well corroborated with the wet-lab observations indicating that MD-guided rational design could lead to discovery of potent AMPs. The effect of the residue substitution on membrane activity was studied in greater detail using killing kinetic analysis. Killing kinetics studies on Gram-positive, negative and human erythrocytes indicated that a single residue change has a drastic effect on the potency of AMPs. An interesting outcome was a switch from monophasic to biphasic death rate constant of Staphylococcus aureus due to a single residue mutation in the peptide. PMID:29527201
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Tanner, Jerome E; Coinçon, Mathieu; Leblond, Valérie; Hu, Jing; Fang, Janey M; Sygusch, Jurgen; Alfieri, Caroline
2015-05-01
Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope. The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of the gp350 interaction with a virus-blocking monoclonal antibody. Immunization with gp350 peptides identified by in silico mapping generated antibodies that cross-react with the EBV gp350 molecule and block recognition of the gp350 molecule by a virus-neutralizing antibody. Through its ability to focus the immune system exclusively on the gp350 sequence important for viral entry, these peptides may form the basis of an EBV vaccine candidate. This strategy would sidestep the production of other irrelevant gp350 antibodies that divert the immune system from generating a protective antiviral response or that impede access to the virus-blocking epitope by protective antibodies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Yuan, Han-Chih; Wu, Keh-Gong; Chen, Chun-Jen; Su, Song-Nan; Shen, Horng-Der; Chen, Yann-Jang; Peng, Ho-Jen
2012-01-01
Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG(4) antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG(1-4) antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. Four major IgE-binding epitopes (peptides 15(-1), 21, 33(-2) and 35(+1), corresponding to amino acids 70-79, 101-110, 159-167 and 172-181) and 5 major IgG(4)-binding epitopes (peptides 15(-1), 30(-2), 33(-2), 35(+1) and 39, corresponding to amino acids 70-79, 144-153, 159-167, 172-181 and 192-200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG(4). Their critical amino acids were all characterized. Major epitopes for human IgG(1) to IgG(4) are almost identical. This is the first study to map the sequential epitopes for human IgE and IgG(4) subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis. Copyright © 2011 S. Karger AG, Basel.
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The influence of solvent on conformational properties of peptides with Aib residue-a DFT study.
Wałęsa, Roksana; Broda, Małgorzata A
2017-11-21
The conformational propensities of the Aib residue on the example of two model peptides Ac-Aib-NHMe (1) and Ac-Aib-NMe 2 (2), were studied by B3LYP and M06-2X functionals, in the gas phase and in the polar solvents. To verify the reliability of selected functionals, we also performed MP2 calculations for the tested molecules in vacuum. Polarizable continuum models (PCM and SMD) were used to estimate the solvent effect. Ramachandran maps were calculated to find all energy minima. Noncovalent intramolecular interactions due to hydrogen-bonds and dipole attractions between carbonyl groups are responsible for the relative stabilities of the conformers. In order to verify the theoretical results, the available conformations of similar X-ray structures from the Cambridge Crystallographic Data Center (CCDC) were analyzed. The results of the calculations show that both derivatives with the Aib residue in the gas phase prefer structures stabilized by intramolecular N-H⋯O hydrogen bonds, i.e., C 5 and C 7 conformations, while polar solvent promotes helical conformation with φ, ψ values equal to +/-60°, +/-40°. In addition, in the case of molecule 2, the helical conformation is the only one available in the polar environment. This result is fully consistent with the X-ray data. Graphical abstract Effect of solvent on the Ramachandran maps of the model peptides with Aib residue.
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Liu, Nai-Yu; Lee, Hsiao-Hui; Chang, Zee-Fen; Tsay, Yeou-Guang
2015-09-10
It has been observed that a modified peptide and its non-modified counterpart, when analyzed with reverse phase liquid chromatography, usually share a very similar elution property [1-3]. Inasmuch as this property is common to many different types of protein modifications, we propose an informatics-based approach, featuring the generation of segmental average mass spectra ((sa)MS), that is capable of locating different types of modified peptides in two-dimensional liquid chromatography-mass spectrometric (LC-MS) data collected for regular protease digests from proteins in gels or solutions. To enable the localization of these peptides in the LC-MS map, we have implemented a set of computer programs, or the (sa)MS package, that perform the needed functions, including generating a complete set of segmental average mass spectra, compiling the peptide inventory from the Sequest/TurboSequest results, searching modified peptide candidates and annotating a tandem mass spectrum for final verification. Using ROCK2 as an example, our programs were applied to identify multiple types of modified peptides, such as phosphorylated and hexosylated ones, which particularly include those peptides that could have been ignored due to their peculiar fragmentation patterns and consequent low search scores. Hence, we demonstrate that, when complemented with peptide search algorithms, our approach and the entailed computer programs can add the sequence information needed for bolstering the confidence of data interpretation by the present analytical platforms and facilitate the mining of protein modification information out of complicated LC-MS/MS data. Copyright © 2015 Elsevier B.V. All rights reserved.
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Advancement and applications of peptide phage display technology in biomedical science.
Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung
2016-01-19
Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.
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Attractors in Sequence Space: Agent-Based Exploration of MHC I Binding Peptides.
Jäger, Natalie; Wisniewska, Joanna M; Hiss, Jan A; Freier, Anja; Losch, Florian O; Walden, Peter; Wrede, Paul; Schneider, Gisbert
2010-01-12
Ant Colony Optimization (ACO) is a meta-heuristic that utilizes a computational analogue of ant trail pheromones to solve combinatorial optimization problems. The size of the ant colony and the representation of the ants' pheromone trails is unique referring to the given optimization problem. In the present study, we employed ACO to generate novel peptides that stabilize MHC I protein on the plasma membrane of a murine lymphoma cell line. A jury of feedforward neural network classifiers served as fitness function for peptide design by ACO. Bioactive murine MHC I H-2K(b) stabilizing as well as nonstabilizing octapeptides were designed, synthesized and tested. These peptides reveal residue motifs that are relevant for MHC I receptor binding. We demonstrate how the performance of the implemented ACO algorithm depends on the colony size and the size of the search space. The actual peptide design process by ACO constitutes a search path in sequence space that can be visualized as trajectories on a self-organizing map (SOM). By projecting the sequence space on a SOM we visualize the convergence of the different solutions that emerge during the optimization process in sequence space. The SOM representation reveals attractors in sequence space for MHC I binding peptides. The combination of ACO and SOM enables systematic peptide optimization. This technique allows for the rational design of various types of bioactive peptides with minimal experimental effort. Here, we demonstrate its successful application to the design of MHC-I binding and nonbinding peptides which exhibit substantial bioactivity in a cell-based assay. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Mapping protease substrates using a biotinylated phage substrate library.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Scholle, M. D.; Kriplani, U.; Pabon, A.
We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobicmore » is the scissile bond.« less
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An open-source computational and data resource to analyze digital maps of immunopeptidomes
Caron, Etienne; Espona, Lucia; Kowalewski, Daniel J; Schuster, Heiko; Ternette, Nicola; Alpízar, Adán; Schittenhelm, Ralf B; Ramarathinam, Sri H; Lindestam Arlehamn, Cecilia S; Chiek Koh, Ching; Gillet, Ludovic C; Rabsteyn, Armin; Navarro, Pedro; Kim, Sangtae; Lam, Henry; Sturm, Theo; Marcilla, Miguel; Sette, Alessandro; Campbell, David S; Deutsch, Eric W; Moritz, Robert L; Purcell, Anthony W; Rammensee, Hans-Georg; Stevanovic, Stefan; Aebersold, Ruedi
2015-01-01
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies. DOI: http://dx.doi.org/10.7554/eLife.07661.001 PMID:26154972
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Cruz, Luis J; Rueda, Felix; Simón, Lorena; Cordobilla, Begoña; Albericio, Fernando; Domingo, Joan C
2014-04-01
To improve the immunological response against tumors, a vaccine based on nanoliposomes targeted to the Fcg-receptor was developed to enhance the immunogenicity of tumor-associated antigens (TAAs). Using human dendritic cells in vitro, a fragment of the TAA NY-ESO-1 combined with a T-helper peptide from the tetanus toxoid encapsulated in nanoliposomes was evaluated. In addition, peptides Palm-IL-1 and MAP-IFN-g were coadministered as adjuvants to enhance the immunological response. Coadministration of Palm-IL-1 or MAP-IFN-g peptide adjuvants and the hybrid NY-ESO-1-tetanus toxoid (soluble or encapsulated in nanoliposomes without targeting) increased immunogenicity. However, the most potent immunological response was obtained when the peptide adjuvants were encapsulated in liposomes targeted to human dendritic cells via the Fc receptor. This targeted vaccine strategy is a promising tool to activate and deliver antigens to dendritic cells, thus improving immunotherapeutic response in situations in which the immune system is frequently compromised, as in advanced cancers.
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Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-β peptide
NASA Astrophysics Data System (ADS)
Cohen, Samuel I. A.; Cukalevski, Risto; Michaels, Thomas C. T.; Šarić, Andela; Törnquist, Mattias; Vendruscolo, Michele; Dobson, Christopher M.; Buell, Alexander K.; Knowles, Tuomas P. J.; Linse, Sara
2018-05-01
Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloid-β peptide (Aβ42), which is associated with Alzheimer's disease. Our results reveal that interactions between monomeric Aβ42 and amyloid fibrils during fibril-dependent secondary nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reaction trajectories, we show that the catalytic efficiency of Aβ42 fibril surfaces results from the enthalpic stabilization of adsorbing peptides in conformations amenable to nucleation, resulting in a dramatic lowering of the activation energy for nucleation.
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Cohen, Samuel I A; Cukalevski, Risto; Michaels, Thomas C T; Šarić, Anđela; Törnquist, Mattias; Vendruscolo, Michele; Dobson, Christopher M; Buell, Alexander K; Knowles, Tuomas P J; Linse, Sara
2018-05-01
Mapping free-energy landscapes has proved to be a powerful tool for studying reaction mechanisms. Many complex biomolecular assembly processes, however, have remained challenging to access using this approach, including the aggregation of peptides and proteins into amyloid fibrils implicated in a range of disorders. Here, we generalize the strategy used to probe free-energy landscapes in protein folding to determine the activation energies and entropies that characterize each of the molecular steps in the aggregation of the amyloid-β peptide (Aβ42), which is associated with Alzheimer's disease. Our results reveal that interactions between monomeric Aβ42 and amyloid fibrils during fibril-dependent secondary nucleation fundamentally reverse the thermodynamic signature of this process relative to primary nucleation, even though both processes generate aggregates from soluble peptides. By mapping the energetic and entropic contributions along the reaction trajectories, we show that the catalytic efficiency of Aβ42 fibril surfaces results from the enthalpic stabilization of adsorbing peptides in conformations amenable to nucleation, resulting in a dramatic lowering of the activation energy for nucleation.
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Wang, Zhouxi; Rejtar, Tomas; Zhou, Zhaohui Sunny; Karger, Barry L.
2010-01-01
In this paper, we have examined two cysteine modifications resulting from sample preparation for protein characterization by MS: (1) a previously observed conversion of cysteine to dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine to alanine. Using model peptides, the conversion of cysteine to dehydroalanine via β-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 °C, pH 7.0– 9.0) without disulfide reduction and alkylation.. Furthermore, the surprising conversion of cysteine to alanine was shown to occur by heating cysteine containing peptides in the presence of a phosphine (TCEP). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to β-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. PMID:20049891
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Barbosa, Eder Alves; Iembo, Tatiane; Martins, Graciella Ribeiro; Silva, Luciano Paulino; Prates, Maura Vianna; Andrade, Alan Carvalho; Bloch, Carlos
2015-11-15
Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior. Copyright © 2015 John Wiley & Sons, Ltd.
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Anumula, K R; Du, P
1999-11-15
Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose. Copyright 1999 Academic Press.
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Oscherwitz, Jon; Yu, Fen; Cease, Kemp B
2010-09-15
The current vaccines for anthrax in the United States and United Kingdom are efficacious in the two most accepted animal models of inhalation anthrax, nonhuman primates and rabbits, but require extensive immunization protocols. We previously demonstrated that a linear determinant in domain 2 of Bacillus anthracis protective Ag (PA) is a potentially important target for an epitope-specific vaccine for anthrax, as Abs specific for this site, referred to as the loop-neutralizing determinant (LND), neutralize lethal toxin in vitro, yet are virtually absent in PA-immunized rabbits. In this study, we evaluated the immunogenicity and protective efficacy in rabbits of multiple antigenic peptides (MAPs) consisting of aa 304-319 from the LND of PA colinearly synthesized at the C terminus (T-B MAP) or N terminus (B-T MAP) with a heterologous T cell epitope from Plasmodium falciparum. Immunogenicity studies demonstrated that both MAPs elicited toxin-neutralizing Ab in rabbits. To evaluate the MAPs as potential anthrax vaccines, we immunized groups of rabbits (n = 7) with each MAP in Freund's adjuvant and then exposed all rabbits to a 200-LD(50) challenge with aerosolized spores of B. anthracis Ames strain. All seven rabbits immunized with the B-T MAP and 89% (six of seven) of rabbits immunized with the T-B MAP survived the spore challenge. Corollary studies with reference sera from human vaccinees immunized with rPA or anthrax vaccine absorbed and nonhuman primates immunized with PA revealed no detectable Ab with specificity for the LND. We conclude that a synthetic peptide vaccine targeting the LND would be a potentially efficacious vaccine for anthrax.
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Pinna, Antonio; Masala, Speranza; Blasetti, Francesco; Maiore, Irene; Cossu, Davide; Paccagnini, Daniela; Mameli, Giuseppe; Sechi, Leonardo A
2014-01-01
MAP3865c, a Mycobacterium avium subspecies paratuberculosis (MAP) cell membrane protein, has a relevant sequence homology with zinc transporter 8 (ZnT8), a beta-cell membrane protein involved in Zn++ transportation. Recently, antibodies recognizing MAP3865c epitopes have been shown to cross-react with ZnT8 in type 1 diabetes patients. The purpose of this study was to detect antibodies against MAP3865c peptides in patients with high-risk proliferative diabetic retinopathy and speculate on whether they may somehow be involved in the pathogenesis of this severe retinal disorder. Blood samples were obtained from 62 type 1 and 80 type 2 diabetes patients with high-risk proliferative diabetic retinopathy and 81 healthy controls. Antibodies against 6 highly immunogenic MAP3865c peptides were detected by indirect ELISA. Type 1 diabetes patients had significantly higher rates of positive antibodies than controls. Conversely, no statistically significant differences were found between type 2 diabetes patients and controls. After categorization of type 1 diabetes patients into two groups, one with positive, the other with negative antibodies, we found that they had similar mean visual acuity (∼ 0.6) and identical rates of vitreous hemorrhage (28.6%). Conversely, Hashimoto's thyroiditis prevalence was 4/13 (30.7%) in the positive antibody group and 1/49 (2%) in the negative antibody group, a statistically significant difference (P = 0.016). This study confirmed that type 1 diabetes patients have significantly higher rates of positive antibodies against MAP/ZnT8 peptides, but failed to find a correlation between the presence of these antibodies and the severity degree of high-risk proliferative diabetic retinopathy. The significantly higher prevalence of Hashimoto's disease among type 1 diabetes patients with positive antibodies might suggest a possible common environmental trigger for these conditions.
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Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P
1996-10-01
An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct epitopes to be identified over ELISA.
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NASA Astrophysics Data System (ADS)
Long, Ying; Wood, Troy D.
2015-01-01
Most enzymatic microreactors for protein digestion are based on trypsin, but proteins with hydrophobic segments may be difficult to digest because of the paucity of Arg and Lys residues. Microreactors based on pepsin, which is less specific than trypsin, can overcome this challenge. Here, an integrated immobilized pepsin microreactor (IPMR)/nanoelectrospray emitter is examined for its potential for peptide mapping. For myoglobin, equivalent sequence coverage is obtained in a thousandth the time of solution digestion with better sequence coverage. While sequence coverage of cytochrome c is lesser than solution in this short duration, more highly-charged peptic peptides are produced and a number of peaks are unidentified at low-resolution, suggesting that high-resolution mass spectrometry is needed to take full advantage of integrated IPMR/nanoelectrospray devices.
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MHC class I-associated peptides derive from selective regions of the human genome.
Pearson, Hillary; Daouda, Tariq; Granados, Diana Paola; Durette, Chantal; Bonneil, Eric; Courcelles, Mathieu; Rodenbrock, Anja; Laverdure, Jean-Philippe; Côté, Caroline; Mader, Sylvie; Lemieux, Sébastien; Thibault, Pierre; Perreault, Claude
2016-12-01
MHC class I-associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology.
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MHC class I–associated peptides derive from selective regions of the human genome
Pearson, Hillary; Granados, Diana Paola; Durette, Chantal; Bonneil, Eric; Courcelles, Mathieu; Rodenbrock, Anja; Laverdure, Jean-Philippe; Côté, Caroline; Thibault, Pierre
2016-01-01
MHC class I–associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology. PMID:27841757
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Lindsay, R M; Stevenson, K J
1976-01-01
1. An anionic and a cationic chymotrypsin (EC 3.4.21.1) were isolated from the pancreas glands of the moose (Alces alces) and elk (Cervus elaphus). The A and B chymotrypsins from each species were purified to homogeneity by (NH4)2SO4 fractionation, affinity chromatography on 4-phenylbutylamine-Sepharose and ion-exchange chromatography on DEAE- and CM-cellulose. 2. The molecular weight and pH optimum of each chymotrypsin were similar to those of the corresponding ox A and B chymotrypsins. 3. The substrate specificities of the chymotrypsins were investigated by digestion of glucagon and the oxidized B chain of insulin. The primary specificity of each chymotrypsin for aromatic amino acid residues was further established by determining the Km and kcat for the hydrolysis of a number of synthetic amino acid ester substrates. 4. The amino acid composition and total number of residues of moose and elk chymotrypsin A were similar to those of ox chymotrypsin A. An even greater similarity was observed among the B chymotrypsins of the three species. 5. The A chymotrypsins of moose and elk were fragmented to their constituent 'A', 'B' and 'C' polypeptide chains by succinylation (3-carboxypropionylation), reduction and alkylation of the native enzymes. In each case, the two major chains ('B' and 'C') were separated and isolated. By comparison of the amino acid compositions of moose, elk and oxy 'B' and 'C' chains, a greater difference was observed among the three A chymotrypsins than was suggested by the amino acid compositions of the native enzymes alone. 6. Peptides were isolated from the disulphide bridge and active-site regions of the A and B chymotrypsins of moose and elk by diagonal peptide-'mapping' techniques. From the amino acid compositions of the isolated peptides (assuming maximum homology) and from a comparison of diagonal peptide 'maps', there was established a high degree of primary-structure identity among the mooae, elk and ox chymotrypsins. Tentative sequences were deduced for the peptides isolated by diagonal peptide 'mapping'. 7. Details of the isolation procedures of the moose and elk chymotrypsins A and B and the amino acid analyses of some peptides obtained by diagonal peptide 'mapping' have been deposited as Supplementary Publication SUP 50064 (27 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5. Images PLATE 1 PMID:949318
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Sheynkman, Gloria M.; Shortreed, Michael R.; Frey, Brian L.; Scalf, Mark; Smith, Lloyd M.
2013-01-01
Each individual carries thousands of non-synonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level in order to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses, due to their absence from the generic databases that are employed for mass spectrometry searching. Here, we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep coverage shotgun MS data obtained from the same sample. This approach enabled detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data, and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs, and found to be comparable in all cases. PMID:24175627
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Mohammadi, Mohammad; Rasaee, Mohammad Javad; Rajabibazl, Masoumeh; Paknejad, Malihe; Zare, Mehrak; Mohammadzadeh, Sara
2007-08-01
PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.
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Bo, Marco; Erre, Gian Luca; Niegowska, Magdalena; Piras, Marco; Taras, Loredana; Longu, Maria Giovanna; Passiu, Giuseppe; Sechi, Leonardo A
2018-01-01
Rheumatoid arthritis (RA) is a chronic disease characterised by a pro-inflammatory cytokines linked erosive joint damage and by humoral and cellular response against a broad range of self-peptides. Molecular mimicry between Epstein-Barr virus (EBV), Mycobacterium avium subsp. paratuberculosis (MAP) and host peptides has long been regarded as an RA pathogenetic mechanism. Using bioinformatic analysis we identified high sequence homology among interferon regulatory factor 5 (IRF5), EBV antigen BOLF1 and MAP antigen MAP_4027. Our objective was to evaluate the presence in sera of RA patients of antibodies (Abs) directed against human homologous IRF5 cross-reacting with BOLF1 and MAP_4027. Frequency of reactivity against IRF5424-434, BOLF1305-320 and MAP_402718-32 was tested by indirect ELISA in sera from 71 RA patients and 60 healthy controls (HCs). RA sera show a remarkable high frequency of reactivity against IRF5424-434 in comparison to HCs (69% vs. 8%; p<0.0001). Similarly, seroreactivity against BOLF1305-320 was more frequently detected in RA sera than in HCs counterpart (58% vs. 8%; p<0.0001). Frequency of Abs against MAP_402718-32 was 17% in RA sera vs. 5% in HCs with a p-value at the threshold level (p<0.051). Prevalence of Abs against at least one of the assessed epitopes reached 72% in RA patients and 15% among HCs. Levels of Abs in RA patients were significantly related to systemic inflammation. IRF5 is a potential autoimmune target of RA. Our results support the hypothesis that EBV and MAP infections may be involved in the pathogenesis of RA, igniting a secondary immune response that cross-reacts against RA self-peptides.
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Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes
Ruwona, Tinashe B; Mcbride, Ryan; Chappel, Rebecca; Head, Steven R; Ordoukhanian, Phillip; Burton, Dennis R.; Law, Mansun
2014-01-01
Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research. PMID:24269751
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A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.
Hong, Qiuting; Ruhaak, L Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual; Lebrilla, Carlito B
2015-12-04
A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
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NASA Astrophysics Data System (ADS)
Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L. C.
2011-12-01
We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D2O and compare with experimental observations.
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Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L C
2011-12-21
We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.
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Choong, Wai-Kok; Lih, Tung-Shing Mamie; Chen, Yu-Ju; Sung, Ting-Yi
2017-12-01
To confirm the existence of missing proteins, we need to identify at least two unique peptides with length of 9-40 amino acids of a missing protein in bottom-up mass-spectrometry-based proteomic experiments. However, an identified unique peptide of the missing protein, even identified with high level of confidence, could possibly coincide with a peptide of a commonly observed protein due to isobaric substitutions, mass modifications, alternative splice isoforms, or single amino acid variants (SAAVs). Besides unique peptides of missing proteins, identified variant peptides (SAAV-containing peptides) could also alternatively map to peptides of other proteins due to the aforementioned issues. Therefore, we conducted a thorough comparative analysis on data sets in PeptideAtlas Tiered Human Integrated Search Proteome (THISP, 2017-03 release), including neXtProt (2017-01 release), to systematically investigate the possibility of unique peptides in missing proteins (PE2-4), unique peptides in dubious proteins, and variant peptides affected by isobaric substitutions, causing doubtful identification results. In this study, we considered 11 isobaric substitutions. From our analysis, we found <5% of the unique peptides of missing proteins and >6% of variant peptides became shared with peptides of PE1 proteins after isobaric substitutions.
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Harden, Bradley J; Nichols, Scott R; Frueh, Dominique P
2014-09-24
Nuclear magnetic resonance (NMR) studies of larger proteins are hampered by difficulties in assigning NMR resonances. Human intervention is typically required to identify NMR signals in 3D spectra, and subsequent procedures depend on the accuracy of this so-called peak picking. We present a method that provides sequential connectivities through correlation maps constructed with covariance NMR, bypassing the need for preliminary peak picking. We introduce two novel techniques to minimize false correlations and merge the information from all original 3D spectra. First, we take spectral derivatives prior to performing covariance to emphasize coincident peak maxima. Second, we multiply covariance maps calculated with different 3D spectra to destroy erroneous sequential correlations. The maps are easy to use and can readily be generated from conventional triple-resonance experiments. Advantages of the method are demonstrated on a 37 kDa nonribosomal peptide synthetase domain subject to spectral overlap.
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Pane, Katia; Durante, Lorenzo; Crescenzi, Orlando; Cafaro, Valeria; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Izzo, Viviana; Di Donato, Alberto; Notomista, Eugenio
2017-04-21
Cationic antimicrobial peptides (CAMPs) are essential components of innate immunity. Here we show that antimicrobial potency of CAMPs is linearly correlated to the product C m H n L where C is the net charge of the peptide, H is a measure of its hydrophobicity and L its length. Exponents m and n define the relative contribution of charge and hydrophobicity to the antimicrobial potency. Very interestingly the values of m and n are strain specific. The ratio n/(m+n) can vary between ca. 0.5 and 1, thus indicating that some strains are sensitive to highly charged peptides, whereas others are particularly susceptible to more hydrophobic peptides. The slope of the regression line describing the correlation "antimicrobial potency"/"C m H n L product" changes from strain to strain indicating that some strains acquired a higher resistance to CAMPs than others. Our analysis provides also an effective computational strategy to identify CAMPs included inside the structure of larger proteins or precursors, which can be defined as "cryptic" CAMPs. We demonstrate that it is not only possible to identify and locate with very good precision the position of cryptic peptides, but also to analyze the internal structure of long CAMPs, thus allowing to draw an accurate map of the molecular determinants of their antimicrobial activity. A spreadsheet, provided in the Supplementary material, allows performing the analysis of protein sequences. Our strategy is also well suited to analyze large pools of sequences, thus significantly improving the identification of new CAMPs and the study of innate immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Cobb, K A; Novotny, M V
1992-01-01
The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.
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Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Marie; Mine, Yoshinori
The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e.,more » A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-{gamma} and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.« less
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Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.
Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki
2017-09-02
Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.
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Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kay, B. K.; Castagnoli, L.; Biosciences Division
This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.
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Mapping of Epitopes Occurring in Bovine α(s1)-Casein Variants by Peptide Microarray Immunoassay.
Lisson, Maria; Erhardt, Georg
2016-01-01
Immunoglobulin E epitope mapping of milk proteins reveals important information about their immunologic properties. Genetic variants of αS1-casein, one of the major allergens in bovine milk, are until now not considered when discussing the allergenic potential. Here we describe the complete procedure to assess the allergenicity of αS1-casein variants B and C, which are frequent in most breeds, starting from milk with identification and purification of casein variants by isoelectric focusing (IEF) and anion-exchange chromatography, followed by in vitro gastrointestinal digestion of the casein variants, identification of the resulting peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), in silico analysis of the variant-specific peptides as allergenic epitopes, and determination of their IgE-binding properties by microarray immunoassay with cow's milk allergic human sera.
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Maiuri, Luigi; Ciacci, Carolina; Ricciardelli, Ida; Vacca, Loredana; Raia, Valeria; Auricchio, Salvatore; Picard, Jean; Osman, Mohamed; Quaratino, Sonia; Londei, Marco
2003-07-05
The adaptive immune system is central to the development of coeliac disease. Adaptive immune responses are, however, controlled by a preceding activation of the innate immune system. We investigated whether gliadin, a protein present in wheat flour, could activate an innate as well as an adaptive immune response in patients with coeliac disease. Duodenal biopsy samples from 42 patients with untreated coeliac disease, 37 treated patients, and 18 controls, were cultured in vitro for 3 h or 24 h, in the presence of either immunodominant gliadin epitopes (p(alpha)-2 and p(alpha)-9) or a non-immunodominant peptide (p31-43) known to induce small intestine damage in coeliac disease. We also incubated biopsy samples from nine untreated and six treated patients with a non-immunodominant peptide for 3 h, before incubation with immunodominant gliadin epitopes. Different combinations of interleukin-15 or signal transduction inhibitors were added to selected incubations. Only the non-immunodominant peptide induced rapid expression of interleukin-15, CD83, cyclo-oxygenase (COX)-2, and CD25 by CD3- cells (p=0.005 vs medium alone) and enterocyte apoptosis (p<0.0001). Only the non-immunodominant peptide induced p38 MAP kinase activation in CD3- cells. Pre-incubation with the non-immunodominant peptide enabled immunodominant epitopes to induce T-cell activation (p=0.001) and enterocyte apoptosis. Inhibition of interleukin-15 or of p38 MAP kinase controlled such activity. A gliadin fragment can activate the innate immune system, affecting the in situ T-cell recognition of dominant gliadin epitopes. Although our findings emphasise the key role of gliadin-specific T cells, they suggest a complex pathogenic situation, and show that inhibition of interleukin-15 or p38 MAP kinase might have the potential to control coeliac disease.
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Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM
2009-01-01
Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584
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Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.
Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A
2015-03-01
The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Mapping Antigenic Motifs in the Trypomastigote Small Surface Antigen from Trypanosoma cruzi
Balouz, Virginia; Cámara, María de los Milagros; Cánepa, Gaspar E.; Carmona, Santiago J.; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán
2015-01-01
The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. PMID:25589551
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Chen, Shun-Li; Wu, Shiaw-Lin; Huang, Li-Juan; Huang, Jia-Bao; Chen, Shu-Hui
2013-06-01
Liquid chromatography-tandem mass spectrometry-based proteomics for peptide mapping and sequencing was used to characterize the marketed monoclonal antibody trastuzumab and compare it with two biosimilar products, mAb A containing D359E and L361M variations at the Fc site and mAb B without variants. Complete sequence coverage (100%) including disulfide linkages, glycosylations and other commonly occurring modifications (i.e., deamidation, oxidation, dehydration and K-clipping) were identified using maps generated from multi-enzyme digestions. In addition to the targeted comparison for the relative populations of targeted modification forms, a non-targeted approach was used to globally compare ion intensities in tryptic maps. The non-targeted comparison provided an extra-dimensional view to examine any possible differences related to variants or modifications. A peptide containing the two variants in mAb A, D359E and L361M, was revealed using the non-targeted comparison of the tryptic maps. In contrast, no significant differences were observed when trastuzumab was self-compared or compared with mAb B. These results were consistent with the data derived from peptide sequencing via collision induced dissociation/electron transfer dissociation. Thus, combined targeted and non-targeted approaches using powerful mass spectrometry-based proteomic tools hold great promise for the structural characterization of biosimilar products. Copyright © 2013 Elsevier B.V. All rights reserved.
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21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune Addison's disease.
Rottembourg, Diane; Deal, Cheri; Lambert, Marion; Mallone, Roberto; Carel, Jean-Claude; Lacroix, André; Caillat-Zucman, Sophie; le Deist, Françoise
2010-12-01
In autoimmune adrenal deficiency, autoantibodies target the 21-hydroxylase (21OH) protein. However, it is presumed that autoreactive T cells, rather than antibodies, are the main effectors of adrenal gland destruction, but their identification is still lacking. We performed a T-cell epitope mapping study using 49 overlapping 20mer peptides covering the 21OH sequence in patients with isolated Addison's disease, Autoimmune Polyendocrine Syndrome 1 and 2. IFNγ ELISPOT responses against these peptides were stronger, broader and more prevalent among patients than in controls, whatever the disease presentation. Five peptides elicited T-cell responses in patients only (68% sensitivity, 100% specificity). Blocking experiments identified IFNγ-producing cells as CD8 T lymphocytes, with two peptides frequently recognized in HLA-B8+ patients and a third one targeted in HLA-B35+ subjects. In particular, the 21OH(431-450) peptide was highly immunodominant, as it was recognized in more than 30% of patients, all carrying the HLA-B8 restriction element. This 21OH(431-450) region contained an EPLARLEL octamer (21OH(431-438)) predicted to bind to HLA-B8 with high affinity. Indeed, circulating EPLARLEL-specific CD8 T cells were detected at significant frequencies in HLA-B8+ patients but not in controls by HLA tetramer staining. This report enlightens disease-specific T-cell biomarkers and epitopes targeted in autoimmune adrenal deficiency. Copyright © 2010 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry
Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less
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Experimental verification of force fields for molecular dynamics simulations using Gly-Pro-Gly-Gly.
Aliev, Abil E; Courtier-Murias, Denis
2010-09-30
Experimental NMR verification of MD simulations using 12 different force fields (AMBER, CHARMM, GROMOS, and OPLS-AA) and 5 different water models has been undertaken to identify reliable MD protocols for structure and dynamics elucidations of small open chain peptides containing Gly and Pro. A conformationally flexible tetrapeptide Gly-Pro-Gly-Gly was selected for NMR (3)J-coupling, chemical shift, and internuclear distance measurements, followed by their calculations using 2 μs long MD simulations in water. In addition, Ramachandran population maps for Pro-2 and Gly-3 residues of GPGG obtained from MD simulations were used for detailed comparisons with similar maps from the protein data bank (PDB) for large number of Gly and Pro residues in proteins. The MD simulations revealed strong dependence of the populations and geometries of preferred backbone and side chain conformations, as well as the time scales of the peptide torsional transitions on the force field used. On the basis of the analysis of the measured and calculated data, AMBER99SB is identified as the most reliable force field for reproducing NMR measured parameters, which are dependent on the peptide backbone and the Pro side chain geometries and dynamics. Ramachandran maps showing the dependence of conformational populations as a function of backbone ϕ/ψ angles for Pro-2 and Gly-3 residues of GPGG from MD simulations using AMBER99SB, AMBER03, and CHARMM were found to resemble similar maps for Gly and Pro residues from the PDB survey. Three force fields (AMBER99, AMBER99ϕ, and AMBER94) showed the least satisfactory agreement with both the solution NMR and the PDB survey data. The poor performance of these force fields is attributed to their propensity to overstabilize helical peptide backbone conformations at the Pro-2 and Gly-3 residues. On the basis of the similarity of the MD and PDB Ramachandran plots, the following sequence of transitions is suggested for the Gly backbone conformation: α(L) ⇆ β(PR) ⇆ β(S) ⇆ β(P) ⇆ α, where backbone secondary structures α(L) and α are associated with helices and turns, β(P) and β(PR) correspond to the left- and right-handed polyproline II structures and β(S) denotes the fully stretched backbone conformation. Compared to the force field dependence, less significant, but noteworthy, variations in the populations of the peptide backbone conformations were observed. For different solvent models considered, a correlation was noted between the number of torsional transitions in GPGG and the water self-diffusion coefficient on using TIP3P, TIP4P, and TIP5P models. In addition to MD results, we also report DFT derived Karplus relationships for Gly and Pro residues using B972 and B3LYP functionals.
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Exploiting proteomic data for genome annotation and gene model validation in Aspergillus niger.
Wright, James C; Sugden, Deana; Francis-McIntyre, Sue; Riba-Garcia, Isabel; Gaskell, Simon J; Grigoriev, Igor V; Baker, Scott E; Beynon, Robert J; Hubbard, Simon J
2009-02-04
Proteomic data is a potentially rich, but arguably unexploited, data source for genome annotation. Peptide identifications from tandem mass spectrometry provide prima facie evidence for gene predictions and can discriminate over a set of candidate gene models. Here we apply this to the recently sequenced Aspergillus niger fungal genome from the Joint Genome Institutes (JGI) and another predicted protein set from another A.niger sequence. Tandem mass spectra (MS/MS) were acquired from 1d gel electrophoresis bands and searched against all available gene models using Average Peptide Scoring (APS) and reverse database searching to produce confident identifications at an acceptable false discovery rate (FDR). 405 identified peptide sequences were mapped to 214 different A.niger genomic loci to which 4093 predicted gene models clustered, 2872 of which contained the mapped peptides. Interestingly, 13 (6%) of these loci either had no preferred predicted gene model or the genome annotators' chosen "best" model for that genomic locus was not found to be the most parsimonious match to the identified peptides. The peptides identified also boosted confidence in predicted gene structures spanning 54 introns from different gene models. This work highlights the potential of integrating experimental proteomics data into genomic annotation pipelines much as expressed sequence tag (EST) data has been. A comparison of the published genome from another strain of A.niger sequenced by DSM showed that a number of the gene models or proteins with proteomics evidence did not occur in both genomes, further highlighting the utility of the method.
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Lerner, R A
1983-02-01
Synthetic vaccines are designed with the help of computer-graphics programs. These displays generated by Arthur J. Olson of the Research Institute of Scripps Clinic show a method whereby parts of a viral protein that are on the surface of a virus, and therefore accessible to antibodies, can be identified. The backbone of the surface domain of the protein on the outer shell of the tomato bushy-stunt virus is displayed (1) on the basis of coordinates determined by Stephen C. Harrison of Harvard University and his colleagues. A single peptide of the protein is picked out in yellow, with the side chains of its component amino acids indicated in atomic detail (2). The peptide is enlarged and a sphere representing a water molecule is displayed (3). The sphere is rolled around the peptide to generate a map of the surface accessible to water (4); it does so, following an algorithm developed by Michael L. Connolly, by placing a dot at each point of its closest contact with the peptide, taking account of the sphere's own van der Waals radius (zone of influence, in effect) and that of each atom of the peptide and the rest of the protein. A similar-dot-surface map is generated to show what parts of the peptide are still accessible to water when three copies of the protein are associated in an array on the surface of the virus (5) and when four such arrays (out of 60) are in position on the outer surface of the virus (6).
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Rai, Durgesh K.; Qian, Shuo; Heller, William T.
2016-08-13
We report that membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine andmore » phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L = 1/500, but membrane thinning results when P/L = 1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. Lastly, the results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.« less
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Rai, Durgesh K; Qian, Shuo; Heller, William T
2016-11-01
Membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine and phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L=1/500, but membrane thinning results when P/L=1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. The results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes. Copyright © 2016 Elsevier B.V. All rights reserved.
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Structural characteristics of Tla products
1985-01-01
Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain. PMID:3875681
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Optimization of Feasibility Stage for Hydrogen/Deuterium Exchange Mass Spectrometry
NASA Astrophysics Data System (ADS)
Hamuro, Yoshitomo; Coales, Stephen J.
2018-03-01
The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity. While a typical HDX-MS project starts with a feasibility stage where the experimental conditions are optimized and the peptide map is generated prior to the HDX study stage, the literature usually reports only the HDX study stage. In this protocol, we describe a few considerations for the initial feasibility stage, more specifically, how to optimize quench conditions, how to tackle the carryover issue, and how to apply the pepsin specificity rule. Two sets of quench conditions are described depending on the presence of disulfide bonds to facilitate the quench condition optimization process. Four protocols are outlined to minimize carryover during the feasibility stage: (1) addition of a detergent to the quench buffer, (2) injection of a detergent or chaotrope to the protease column after each sample injection, (3) back-flushing of the trap column and the analytical column with a new plumbing configuration, and (4) use of PEEK (or PEEK coated) frits instead of stainless steel frits for the columns. The application of the pepsin specificity rule after peptide map generation and not before peptide map generation is suggested. The rule can be used not only to remove falsely identified peptides, but also to check the sample purity. A well-optimized HDX-MS feasibility stage makes subsequent HDX study stage smoother and the resulting HDX data more reliable. [Figure not available: see fulltext.
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A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364
DOE Office of Scientific and Technical Information (OSTI.GOV)
Littrell, BobbiJo R
The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm.more » Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.« less
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Shteynberg, David; Deutsch, Eric W.; Lam, Henry; Eng, Jimmy K.; Sun, Zhi; Tasman, Natalie; Mendoza, Luis; Moritz, Robert L.; Aebersold, Ruedi; Nesvizhskii, Alexey I.
2011-01-01
The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets. PMID:21876204
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Artificial-epitope mapping for CK-MB assay.
Tai, Dar-Fu; Ho, Yi-Fang; Wu, Cheng-Hsin; Lin, Tzu-Chieh; Lu, Kuo-Hao; Lin, Kun-Shian
2011-06-07
A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.
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Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin
NASA Astrophysics Data System (ADS)
Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter
1997-12-01
In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.
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Subunit mass analysis for monitoring antibody oxidation.
Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping
2017-04-01
Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.
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The neXtProt peptide uniqueness checker: a tool for the proteomics community.
Schaeffer, Mathieu; Gateau, Alain; Teixeira, Daniel; Michel, Pierre-André; Zahn-Zabal, Monique; Lane, Lydie
2017-11-01
The neXtProt peptide uniqueness checker allows scientists to define which peptides can be used to validate the existence of human proteins, i.e. map uniquely versus multiply to human protein sequences taking into account isobaric substitutions, alternative splicing and single amino acid variants. The pepx program is available at https://github.com/calipho-sib/pepx and can be launched from the command line or through a cgi web interface. Indexing requires a sequence file in FASTA format. The peptide uniqueness checker tool is freely available on the web at https://www.nextprot.org/tools/peptide-uniqueness-checker and from the neXtProt API at https://api.nextprot.org/. lydie.lane@sib.swiss. © The Author(s) 2017. Published by Oxford University Press.
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Candreva, Ángela María; Ferrer-Navarro, Mario; Bronsoms, Silvia; Quiroga, Alejandra; Curciarello, Renata; Cauerhff, Ana; Petruccelli, Silvana; Docena, Guillermo Horacio; Trejo, Sebastián Alejandro
2017-08-01
Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOF MS analysis. On a second approach, the peptide mixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease
Signorino, Giacomo; Arnaboldi, Paul M.; Petzke, Mary M.
2014-01-01
Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease. PMID:24623628
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Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.
Periat, Aurélie; Krull, Ira S; Guillarme, Davy
2015-02-01
This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Peabody, David S.; Chackerian, Bryce; Ashley, Carlee
The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referredmore » to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.« less
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A new genome-mining tool redefines the lasso peptide biosynthetic landscape
Tietz, Jonathan I.; Schwalen, Christopher J.; Patel, Parth S.; Maxson, Tucker; Blair, Patricia M.; Tai, Hua-Chia; Zakai, Uzma I.; Mitchell, Douglas A.
2016-01-01
Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are attractive for genome-driven discovery and re-engineering, but limitations in bioinformatic methods and exponentially increasing genomic data make large-scale mining difficult. We report RODEO (Rapid ORF Description and Evaluation Online), which combines hidden Markov model-based analysis, heuristic scoring, and machine learning to identify biosynthetic gene clusters and predict RiPP precursor peptides. We initially focused on lasso peptides, which display intriguing physiochemical properties and bioactivities, but their hypervariability renders them challenging prospects for automated mining. Our approach yielded the most comprehensive mapping of lasso peptide space, revealing >1,300 compounds. We characterized the structures and bioactivities of six lasso peptides, prioritized based on predicted structural novelty, including an unprecedented handcuff-like topology and another with a citrulline modification exceptionally rare among bacteria. These combined insights significantly expand the knowledge of lasso peptides, and more broadly, provide a framework for future genome-mining efforts. PMID:28244986
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Yamaguchi, Hideto; Hirakura, Yutaka; Shirai, Hiroki; Mimura, Hisashi; Toyo'oka, Toshimasa
2011-06-01
The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control. Copyright © 2011 Elsevier B.V. All rights reserved.
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Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.
Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern
2014-04-08
Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Characterization of fiber-forming peptides and proteins by means of atomic force microscopy.
Creasey, Rhiannon G; Gibson, Christopher T; Voelcker, Nicolas H
2012-05-01
The atomic force microscope (AFM) is widely used in biological sciences due to its ability to perform imaging experiments at high resolution in a physiological environment, without special sample preparation such as fixation or staining. AFM is unique, in that it allows single molecule information of mechanical properties and molecular recognition to be gathered. This review sets out to identify methodological applications of AFM for characterization of fiber-forming proteins and peptides. The basics of AFM operation are detailed, with in-depth information for any life scientist to get a grasp on AFM capabilities. It also briefly describes antibody recognition imaging and mapping of nanomechanical properties on biological samples. Subsequently, examples of AFM application to fiber-forming natural proteins, and fiber-forming synthetic peptides are given. Here, AFM is used primarily for structural characterization of fibers in combination with other techniques, such as circular dichroism and fluorescence spectroscopy. More recent developments in antibody recognition imaging to identify constituents of protein fibers formed in human disease are explored. This review, as a whole, seeks to encourage the life scientists dealing with protein aggregation phenomena to consider AFM as a part of their research toolkit, by highlighting the manifold capabilities of this technique.
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Exploiting proteomic data for genome annotation and gene model validation in Aspergillus niger
Wright, James C; Sugden, Deana; Francis-McIntyre, Sue; Riba-Garcia, Isabel; Gaskell, Simon J; Grigoriev, Igor V; Baker, Scott E; Beynon, Robert J; Hubbard, Simon J
2009-01-01
Background Proteomic data is a potentially rich, but arguably unexploited, data source for genome annotation. Peptide identifications from tandem mass spectrometry provide prima facie evidence for gene predictions and can discriminate over a set of candidate gene models. Here we apply this to the recently sequenced Aspergillus niger fungal genome from the Joint Genome Institutes (JGI) and another predicted protein set from another A.niger sequence. Tandem mass spectra (MS/MS) were acquired from 1d gel electrophoresis bands and searched against all available gene models using Average Peptide Scoring (APS) and reverse database searching to produce confident identifications at an acceptable false discovery rate (FDR). Results 405 identified peptide sequences were mapped to 214 different A.niger genomic loci to which 4093 predicted gene models clustered, 2872 of which contained the mapped peptides. Interestingly, 13 (6%) of these loci either had no preferred predicted gene model or the genome annotators' chosen "best" model for that genomic locus was not found to be the most parsimonious match to the identified peptides. The peptides identified also boosted confidence in predicted gene structures spanning 54 introns from different gene models. Conclusion This work highlights the potential of integrating experimental proteomics data into genomic annotation pipelines much as expressed sequence tag (EST) data has been. A comparison of the published genome from another strain of A.niger sequenced by DSM showed that a number of the gene models or proteins with proteomics evidence did not occur in both genomes, further highlighting the utility of the method. PMID:19193216
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Computer simulation of the active site of human serum cholinesterase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kefang Jiao; Song Li; Zhengzheng Lu
1996-12-31
The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less
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Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel
Veitia, R; David, S; Barbier, P; Vantard, M; Gounon, P; Bissery, M C; Fellous, A
2000-01-01
We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the intensity of the immunostaining is lower. However, anti α-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel. © 2000 Cancer Research Campaign PMID:10945505
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Using peptide array to identify binding motifs and interaction networks for modular domains.
Li, Shawn S-C; Wu, Chenggang
2009-01-01
Specific protein-protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein-protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein-protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein-protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.
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Epitope mapping: the first step in developing epitope-based vaccines.
Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael
2007-01-01
Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have been developed, such as Mapitope, which has recently been found to be effective in mapping conformational discontinuous epitopes. The pros and cons of various approaches towards epitope mapping are also discussed.
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Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F
2011-01-01
The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.
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Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.
2011-01-01
The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532
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2016-01-01
The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ1–40 and Aβ1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ1–42 compared to that of Aβ1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ1–40 and Aβ1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ1–40 and Aβ1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings (3JHNHα, 3JC′C′, 3JC′Hα, 1JHαCα, 2JNCα, and 1JNCα) recorded for Aβ1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process. PMID:26780756
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Roche, Julien; Shen, Yang; Lee, Jung Ho; Ying, Jinfa; Bax, Ad
2016-02-09
The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ(1-40) and Aβ(1-42) into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ(1-42) compared to that of Aβ(1-40) are poorly understood. To explore in detail the structural propensity of the monomeric Aβ(1-40) and Aβ(1-42) peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and J couplings. Systematic comparisons show that at neutral pH the Aβ(1-40) and Aβ(1-42) peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone J couplings ((3)JHNHα, (3)JC'C', (3)JC'Hα, (1)JHαCα, (2)JNCα, and (1)JNCα) recorded for Aβ(1-40) were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for αL at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D2O over H2O, particularly pronounced for Aβ(1-42), suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process.
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Wolski, Witold E; Lalowski, Maciej; Jungblut, Peter; Reinert, Knut
2005-01-01
Background Peptide Mass Fingerprinting (PMF) is a widely used mass spectrometry (MS) method of analysis of proteins and peptides. It relies on the comparison between experimentally determined and theoretical mass spectra. The PMF process requires calibration, usually performed with external or internal calibrants of known molecular masses. Results We have introduced two novel MS calibration methods. The first method utilises the local similarity of peptide maps generated after separation of complex protein samples by two-dimensional gel electrophoresis. It computes a multiple peak-list alignment of the data set using a modified Minimum Spanning Tree (MST) algorithm. The second method exploits the idea that hundreds of MS samples are measured in parallel on one sample support. It improves the calibration coefficients by applying a two-dimensional Thin Plate Splines (TPS) smoothing algorithm. We studied the novel calibration methods utilising data generated by three different MALDI-TOF-MS instruments. We demonstrate that a PMF data set can be calibrated without resorting to external or relying on widely occurring internal calibrants. The methods developed here were implemented in R and are part of the BioConductor package mscalib available from . Conclusion The MST calibration algorithm is well suited to calibrate MS spectra of protein samples resulting from two-dimensional gel electrophoretic separation. The TPS based calibration algorithm might be used to correct systematic mass measurement errors observed for large MS sample supports. As compared to other methods, our combined MS spectra calibration strategy increases the peptide/protein identification rate by an additional 5 – 15%. PMID:16102175
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Loebel, Madlen; Eckey, Maren; Sotzny, Franziska; Hahn, Elisabeth; Bauer, Sandra; Grabowski, Patricia; Zerweck, Johannes; Holenya, Pavlo; Hanitsch, Leif G; Wittke, Kirsten; Borchmann, Peter; Rüffer, Jens-Ulrich; Hiepe, Falk; Ruprecht, Klemens; Behrends, Uta; Meindl, Carola; Volk, Hans-Dieter; Reimer, Ulf; Scheibenbogen, Carmen
2017-01-01
Epstein-Barr-Virus (EBV) plays an important role as trigger or cofactor for various autoimmune diseases. In a subset of patients with Chronic Fatigue Syndrome (CFS) disease starts with infectious mononucleosis as late primary EBV-infection, whereby altered levels of EBV-specific antibodies can be observed in another subset of patients. We performed a comprehensive mapping of the IgG response against EBV comparing 50 healthy controls with 92 CFS patients using a microarray platform. Patients with multiple sclerosis (MS), systemic lupus erythematosus (SLE) and cancer-related fatigue served as controls. 3054 overlapping peptides were synthesised as 15-mers from 14 different EBV proteins. Array data was validated by ELISA for selected peptides. Prevalence of EBV serotypes was determined by qPCR from throat washing samples. EBV type 1 infections were found in patients and controls. EBV seroarray profiles between healthy controls and CFS were less divergent than that observed for MS or SLE. We found significantly enhanced IgG responses to several EBNA-6 peptides containing a repeat sequence in CFS patients compared to controls. EBNA-6 peptide IgG responses correlated well with EBNA-6 protein responses. The EBNA-6 repeat region showed sequence homologies to various human proteins. Patients with CFS had a quite similar EBV IgG antibody response pattern as healthy controls. Enhanced IgG reactivity against an EBNA-6 repeat sequence and against EBNA-6 protein is found in CFS patients. Homologous sequences of various human proteins with this EBNA-6 repeat sequence might be potential targets for antigenic mimicry.
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Exposing hidden alternative backbone conformations in X-ray crystallography using qFit
Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; ...
2015-10-27
Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less
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Validation of molecularly imprinted polymers for side chain selective phosphopeptide enrichment.
Chen, Jing; Shinde, Sudhirkumar; Subedi, Prabal; Wierzbicka, Celina; Sellergren, Börje; Helling, Stefan; Marcus, Katrin
2016-11-04
Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO 2 ) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO 2 enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO 2 failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pY- peptides. Copyright © 2016 Elsevier B.V. All rights reserved.
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Tachibana, K; Marquardt, H; Yokoya, S; Friesen, H G
1988-10-01
We have reported that the secretion of at least 17 distinct peptides [including rat (rGH)] GH by cultured rat pituitary cells was stimulated by GH-releasing hormone and inhibited by somatostatin, when analyzed by two-dimensional polyacrylamide gel electrophoresis. Three of these peptides (no. 23, 24, and 25) were not rGH immunoreactive. In order to determine whether these three peptides are fragments, degradation products or posttranscriptionally modified forms of rGH, rGH and peptide no. 23 were characterized structurally. From partial peptide maps of rGH and peptide no. 23 by V8 protease or chymotrypsin, it appeared that these peptides were not related to each other. By N-terminal microsequencing of two-dimensional polyacrylamide gel electrophoresis purified peptide, we have obtained the sequence of 24 N-terminal amino acid residues of peptide no. 23. This sequence has no significant homology with rGH or any other reported protein sequence. Antiserum was generated against a synthetic oligopeptide corresponding to amino acid residues 3-24 of peptide no. 23. The antiserum cross-reacted with peptides no. 23, 24, and 25 upon Western blot analysis. These results indicate that peptide no. 23 has a novel structure unrelated to other pituitary hormones. Since its secretion is influenced by GH-releasing hormone and somatostatin, peptide no. 23 may represent a previously unrecognized structurally unique growth factor.
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Wolf, Amaya I.; Mozdzanowska, Krystyna; Williams, Katie L.; Singer, David; Richter, Monique; Hoffmann, Ralf; Caton, Andrew J.; Otvos, Laszlo; Erikson, Jan
2011-01-01
Background The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains. Methodology/Principal Findings Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs. Conclusion/Significance Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population. PMID:22180783
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Wolf, Amaya I; Mozdzanowska, Krystyna; Williams, Katie L; Singer, David; Richter, Monique; Hoffmann, Ralf; Caton, Andrew J; Otvos, Laszlo; Erikson, Jan
2011-01-01
The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains. Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs. Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.
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Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics*
Guo, Xiaofeng; Trudgian, David C.; Lemoff, Andrew; Yadavalli, Sivaramakrishna; Mirzaei, Hamid
2014-01-01
Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases. PMID:24696503
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Protein Inference from the Integration of Tandem MS Data and Interactome Networks.
Zhong, Jiancheng; Wang, Jianxing; Ding, Xiaojun; Zhang, Zhen; Li, Min; Wu, Fang-Xiang; Pan, Yi
2017-01-01
Since proteins are digested into a mixture of peptides in the preprocessing step of tandem mass spectrometry (MS), it is difficult to determine which specific protein a shared peptide belongs to. In recent studies, besides tandem MS data and peptide identification information, some other information is exploited to infer proteins. Different from the methods which first use only tandem MS data to infer proteins and then use network information to refine them, this study proposes a protein inference method named TMSIN, which uses interactome networks directly. As two interacting proteins should co-exist, it is reasonable to assume that if one of the interacting proteins is confidently inferred in a sample, its interacting partners should have a high probability in the same sample, too. Therefore, we can use the neighborhood information of a protein in an interactome network to adjust the probability that the shared peptide belongs to the protein. In TMSIN, a multi-weighted graph is constructed by incorporating the bipartite graph with interactome network information, where the bipartite graph is built with the peptide identification information. Based on multi-weighted graphs, TMSIN adopts an iterative workflow to infer proteins. At each iterative step, the probability that a shared peptide belongs to a specific protein is calculated by using the Bayes' law based on the neighbor protein support scores of each protein which are mapped by the shared peptides. We carried out experiments on yeast data and human data to evaluate the performance of TMSIN in terms of ROC, q-value, and accuracy. The experimental results show that AUC scores yielded by TMSIN are 0.742 and 0.874 in yeast dataset and human dataset, respectively, and TMSIN yields the maximum number of true positives when q-value less than or equal to 0.05. The overlap analysis shows that TMSIN is an effective complementary approach for protein inference.
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Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen
2016-01-01
In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.
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Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen
2016-01-01
In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300
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Generative Topographic Mapping of Conformational Space.
Horvath, Dragos; Baskin, Igor; Marcou, Gilles; Varnek, Alexandre
2017-10-01
Herein, Generative Topographic Mapping (GTM) was challenged to produce planar projections of the high-dimensional conformational space of complex molecules (the 1LE1 peptide). GTM is a probability-based mapping strategy, and its capacity to support property prediction models serves to objectively assess map quality (in terms of regression statistics). The properties to predict were total, non-bonded and contact energies, surface area and fingerprint darkness. Map building and selection was controlled by a previously introduced evolutionary strategy allowed to choose the best-suited conformational descriptors, options including classical terms and novel atom-centric autocorrellograms. The latter condensate interatomic distance patterns into descriptors of rather low dimensionality, yet precise enough to differentiate between close favorable contacts and atom clashes. A subset of 20 K conformers of the 1LE1 peptide, randomly selected from a pool of 2 M geometries (generated by the S4MPLE tool) was employed for map building and cross-validation of property regression models. The GTM build-up challenge reached robust three-fold cross-validated determination coefficients of Q 2 =0.7…0.8, for all modeled properties. Mapping of the full 2 M conformer set produced intuitive and information-rich property landscapes. Functional and folding subspaces appear as well-separated zones, even though RMSD with respect to the PDB structure was never used as a selection criterion of the maps. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J
1988-12-01
Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.
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Shikata, Y; Ohe, H; Mano, N; Kuwada, M; Asakawa, N
1998-06-01
The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.
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Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting.
Vaughan, Robert C; Kao, C Cheng
2015-01-01
RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein-RNA contacts within virions.
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Peptide reranking with protein-peptide correspondence and precursor peak intensity information.
Yang, Chao; He, Zengyou; Yang, Can; Yu, Weichuan
2012-01-01
Searching tandem mass spectra against a protein database has been a mainstream method for peptide identification. Improving peptide identification results by ranking true Peptide-Spectrum Matches (PSMs) over their false counterparts leads to the development of various reranking algorithms. In peptide reranking, discriminative information is essential to distinguish true PSMs from false PSMs. Generally, most peptide reranking methods obtain discriminative information directly from database search scores or by training machine learning models. Information in the protein database and MS1 spectra (i.e., single stage MS spectra) is ignored. In this paper, we propose to use information in the protein database and MS1 spectra to rerank peptide identification results. To quantitatively analyze their effects to peptide reranking results, three peptide reranking methods are proposed: PPMRanker, PPIRanker, and MIRanker. PPMRanker only uses Protein-Peptide Map (PPM) information from the protein database, PPIRanker only uses Precursor Peak Intensity (PPI) information, and MIRanker employs both PPM information and PPI information. According to our experiments on a standard protein mixture data set, a human data set and a mouse data set, PPMRanker and MIRanker achieve better peptide reranking results than PetideProphet, PeptideProphet+NSP (number of sibling peptides) and a score regularization method SRPI. The source codes of PPMRanker, PPIRanker, and MIRanker, and all supplementary documents are available at our website: http://bioinformatics.ust.hk/pepreranking/. Alternatively, these documents can also be downloaded from: http://sourceforge.net/projects/pepreranking/.
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Sun, Yuanxia; Hayakawa, Shigeru; Ogawa, Masahiro; Izumori, Ken
2005-12-28
Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.
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NASA Astrophysics Data System (ADS)
Bereau, Tristan; Wang, Zun-Jing; Deserno, Markus
2014-03-01
Interfacial systems are at the core of fascinating phenomena in many disciplines, such as biochemistry, soft-matter physics, and food science. However, the parametrization of accurate, reliable, and consistent coarse-grained (CG) models for systems at interfaces remains a challenging endeavor. In the present work, we explore to what extent two independently developed solvent-free CG models of peptides and lipids—of different mapping schemes, parametrization methods, target functions, and validation criteria—can be combined by only tuning the cross-interactions. Our results show that the cross-parametrization can reproduce a number of structural properties of membrane peptides (for example, tilt and hydrophobic mismatch), in agreement with existing peptide-lipid CG force fields. We find encouraging results for two challenging biophysical problems: (i) membrane pore formation mediated by the cooperative action of several antimicrobial peptides, and (ii) the insertion and folding of the helix-forming peptide WALP23 in the membrane.
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The use of chimeric vimentin citrullinated peptides for the diagnosis of rheumatoid arthritis.
Malakoutikhah, Morteza; Gómara, María J; Gómez-Puerta, José A; Sanmartí, Raimon; Haro, Isabel
2011-11-10
Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and, in many cases, destruction of the joints. To prevent progressive and irreversible structural damage, early diagnosis of RA is of paramount importance. The present study addresses the search of new RA citrullinated antigens that could supplement or complement diagnostic/prognostic existing tests. With this aim, the epitope anticitrullinated vimentin antibody response was mapped using synthetic peptides. To improve the sensitivity/specificity balance, a vimentin peptide that was selected, and its cyclic analogue, were combined with fibrin- and filaggrin-related peptides to render chimeric peptides. Our findings highlight the putative application of these chimeric peptides for the design of RA diagnosis systems and imply that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported here (fibrin, vimentin, filaggrin) has a specific utility in the identification of a particular subset of RA patients.
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Wasslen, Karl V; Tan, Le Hoa; Manthorpe, Jeffrey M; Smith, Jeffrey C
2014-04-01
Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Webb-Robertson, Bobbie-Jo M.
Accurate identification of peptides is a current challenge in mass spectrometry (MS) based proteomics. The standard approach uses a search routine to compare tandem mass spectra to a database of peptides associated with the target organism. These database search routines yield multiple metrics associated with the quality of the mapping of the experimental spectrum to the theoretical spectrum of a peptide. The structure of these results make separating correct from false identifications difficult and has created a false identification problem. Statistical confidence scores are an approach to battle this false positive problem that has led to significant improvements in peptidemore » identification. We have shown that machine learning, specifically support vector machine (SVM), is an effective approach to separating true peptide identifications from false ones. The SVM-based peptide statistical scoring method transforms a peptide into a vector representation based on database search metrics to train and validate the SVM. In practice, following the database search routine, a peptides is denoted in its vector representation and the SVM generates a single statistical score that is then used to classify presence or absence in the sample« less
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The energy landscape of a selective tumor-homing pentapeptide
Zanuy, David; Flores-Ortega, Alejandra; Casanovas, Jordi; Curco, David; Nussinov, Ruth; Aleman, Carlos
2009-01-01
Recently, a potentially powerful strategy based on the of phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified Simulated Annealing combined with Molecular Dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu and Lys, which induces a β-turn motif in the backbone observed in all simulated CREKA systems. PMID:18588341
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Bringing the fathead minnow into the genomic era | Science ...
The fathead minnow is a well-established ecotoxicological model organism that has been widely used for regulatory ecotoxicity testing and research for over a half century. While a large amount of molecular information has been gathered on the fathead minnow over the years, the lack of genomic sequence data has limited the utility of the fathead minnow for certain applications. To address this limitation, high-throughput Illumina sequencing technology was employed to sequence the fathead minnow genome. Approximately 100X coverage was achieved by sequencing several libraries of paired-end reads with differing genome insert sizes. Two draft genome assemblies were generated using the SOAPdenovo and String Graph Assembler (SGA) methods, respectively. When these were compared, the SOAPdenovo assembly had a higher scaffold N50 value of 60.4 kbp versus 15.4 kbp, and it also performed better in a Core Eukaryotic Genes Mapping Analysis (CEGMA), mapping 91% versus 67% of genes. As such, this assembly was selected for further development and annotation. The foundation for genome annotation was generated using AUGUSTUS, an ab initio method for gene prediction. A total of 43,345 potential coding sequences were predicted on the genome assembly. These predicted sequences were translated to peptides and queried in a BLAST search against all vertebrates, with 28,290 of these sequences corresponding to zebrafish peptides and 5,242 producing no significant alignments. Additional ty
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Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.
2014-01-01
Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411
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Ajitha, V S; Muraleedharan, D
2005-03-01
Female sex pheromone production in certain moth species have been shown to be regulated by a cephalic endocrine peptidic factor: pheromone biosynthesis activating neuropeptide (PBAN), having 33 amino acid residues. Antisera against synthetic Heliothis zea-PBAN were developed. Using these polyclonals, immunoreactivity was mapped in the nervous system of Achaea janata. Three distinct groups of immunopositive secretory neurons were identified in the suboesophageal ganglion; and immunoreactivity was observed in the corpora cardiaca, thoracic and in the abdominal ganglia. From about 6000 brain sub-oesophageal ganglion complexes, the neuropeptide was isolated; and purified sequentially by Sep-pak and reversed phase high performance liquid chromatographic methods. Identity of purified PBAN fraction was confirmed with polyclonal antibody by immunoblotting. Molecular mass of the isolated peptide was determined by matrix-assisted laser desorption/ionization mass spectrometry, and was found to be 3900 Da, same as that of known H. zea-PBAN. Radiochemical bioassay confirmed the pheromonotropic effect of the isolated neuropeptide in this insect.
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Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L
2006-01-01
Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.
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Bowen, Elizabeth J.; Schmidt, Thomas W.; Firm, Christina S.; Russo, Andrew F.; Durham, Paul L.
2006-01-01
Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factorα (TNFα). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNFα stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNFα caused a coordinate increase in CGRP promoter activity. TNFα treatment activated the transcription factor NF-κB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNFα induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels. PMID:16277606
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HIPdb: a database of experimentally validated HIV inhibiting peptides.
Qureshi, Abid; Thakur, Nishant; Kumar, Manoj
2013-01-01
Besides antiretroviral drugs, peptides have also demonstrated potential to inhibit the Human immunodeficiency virus (HIV). For example, T20 has been discovered to effectively block the HIV entry and was approved by the FDA as a novel anti-HIV peptide (AHP). We have collated all experimental information on AHPs at a single platform. HIPdb is a manually curated database of experimentally verified HIV inhibiting peptides targeting various steps or proteins involved in the life cycle of HIV e.g. fusion, integration, reverse transcription etc. This database provides experimental information of 981 peptides. These are of varying length obtained from natural as well as synthetic sources and tested on different cell lines. Important fields included are peptide sequence, length, source, target, cell line, inhibition/IC(50), assay and reference. The database provides user friendly browse, search, sort and filter options. It also contains useful services like BLAST and 'Map' for alignment with user provided sequences. In addition, predicted structure and physicochemical properties of the peptides are also included. HIPdb database is freely available at http://crdd.osdd.net/servers/hipdb. Comprehensive information of this database will be helpful in selecting/designing effective anti-HIV peptides. Thus it may prove a useful resource to researchers for peptide based therapeutics development.
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Atrial Natriuretic Peptide Frameshift Mutation in Familial Atrial Fibrillation
Hodgson-Zingman, Denice M.; Karst, Margaret L.; Zingman, Leonid V.; Heublein, Denise M.; Darbar, Dawood; Herron, Kathleen J.; Ballew, Jeffrey D.; de Andrade, Mariza; Burnett, John C.; Olson, Timothy M.
2008-01-01
Summary Atrial fibrillation is a common arrhythmia that is hereditary in a small subgroup of patients. In a family with 11 clinically affected members, we mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide–cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability. PMID:18614783
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Zhao, Cheng; Trudeau, Beth; Xie, Helen; Prostko, John; Fishpaugh, Jeffrey; Ramsay, Carol
2014-06-01
The absolute quantitation of the targeted protein using MS provides a promising method to evaluate/verify biomarkers used in clinical diagnostics. In this study, a cardiac biomarker, troponin I (TnI), was used as a model protein for method development. The epitope peptide of TnI was characterized by epitope excision followed with LC/MS/MS method and acted as the surrogate peptide for the targeted protein quantitation. The MRM-based MS assay using a stable internal standard that improved the selectivity, specificity, and sensitivity of the protein quantitation. Also, plasma albumin depletion and affinity enrichment of TnI by anti-TnI mAb-coated microparticles reduced the sample complexity, enhanced the dynamic range, and further improved the detecting sensitivity of the targeted protein in the biological matrix. Therefore, quantitation of TnI, a low abundant protein in human plasma, has demonstrated the applicability of the targeted protein quantitation strategy through its epitope peptide determined by epitope mapping method. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Paleari, Renata; Caruso, Donatella; Giavarini, Flavio; Colzani, Carlo; Brunati, Pietro; Mosca, Andrea
2012-01-01
We report the first observation of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] in a Caucasian family and the first case of this variant to be found in association with Hb S [β6(A3)Glu→Val, GAG>GTG]. The proband was a healthy 4-year-old Italian boy. His chromatographic hemoglobin (Hb) pattern showed an abnormal peak having the typical retention time of Hb S (25.6% ), a second abnormal peak eluted soon after (13.6%) and a third minor peak eluted at the end of the run (6.5%). Identification of Hb variants were performed by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Two abnormal peptides at m/z 765.1 and 922 were found, corresponding to the αT-4 and βT-1 peptides characteristic for Hb G-Honolulu and Hb S, respectively. The third minor abnormal peak presumably corresponded to the hybrid molecule (α(G-Honolulu)/β(S)). The concomitant presence of Hb G-Honolulu and Hb S does not seem to produce any relevant clinical manifestation.
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SH3 interactome conserves general function over specific form
Xin, Xiaofeng; Gfeller, David; Cheng, Jackie; Tonikian, Raffi; Sun, Lin; Guo, Ailan; Lopez, Lianet; Pavlenco, Alevtina; Akintobi, Adenrele; Zhang, Yingnan; Rual, Jean-François; Currell, Bridget; Seshagiri, Somasekar; Hao, Tong; Yang, Xinping; Shen, Yun A; Salehi-Ashtiani, Kourosh; Li, Jingjing; Cheng, Aaron T; Bouamalay, Dryden; Lugari, Adrien; Hill, David E; Grimes, Mark L; Drubin, David G; Grant, Barth D; Vidal, Marc; Boone, Charles; Sidhu, Sachdev S; Bader, Gary D
2013-01-01
Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form. PMID:23549480
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Zhao, Fen; Yu, Yonghui; Liu, Wei; Zhang, Jian; Liu, Xinqi; Liu, Lingying; Yin, Huinan
2018-03-21
This article describes results of the effect of dietary supplementation with small molecular weight soybean protein-derived peptides on major rat burn injury-induced muscle atrophy. As protein nutrients have been previously implicated to play an important role in improving burn injury outcomes, optimized more readily absorbed small molecular weight soybean protein-derived peptides were evaluated. Thus, the quantity, sodium dodecyl sulfate polyacrylamide-gel electrophoresis patterns, molecular weight distribution, and composition of amino acids of the prepared peptides were analyzed, and a major full-thickness 30% total body surface area burn-injury rat model was utilized to assess the impact of supplementation with soybean protein-derived peptides on initial systemic inflammatory responses as measured by interferon-gamma (IFN-γ), chemokine (C-C motif) ligand 2 (CCL2, also known as MCP-1), chemokine (C-C motif) ligand 7 (CCL7, also known as MCP-3), and generation of muscle atrophy as measured by tibialis anterior muscle (TAM) weight relative to total body weight. Induction of burn injury-induced muscle atrophy ubiquitin-proteasome system (UPS) signaling pathways in effected muscle tissues was determined by Western blot protein expression measurements of E3 ubiquitin-protein ligase TRIM-63 (TRIM63, also known as MuRF1) and F-box only protein 32 (FBXO32, also known as atrogin-1 or MAFbx). In addition, induction of burn injury-induced autophagy signaling pathways associated with muscle atrophy in effected muscle tissues was assessed by immunohistochemical analysis as measured by microtubule-associated proteins 1 light chain 3 (MAP1LC3, or commonly abbreviated as LC3) and beclin-1 (BECN1) expression, as well as relative induction of cytoplasmic-liberated form of MAP1LC3 (LC3-I) and phagophore and autophagosome membrane-bound form of MAP1LC3 (LC3-II), and BECN1 protein expression by Western blot analysis. Nutrient supplementation with small molecular weight soybean protein-derived peptides resulted a significant reduction in burn injury-induced inflammatory markers, muscle atrophy, induction of TRIM63 and FBXO32 muscle atrophy signaling pathways, and induction of autophagy signaling pathways LC3 and BECN1 associated with muscle atrophy. These results implicated that small molecular weight soybean-derived peptides dietary supplementation could be used as an adjunct therapy in burn injury management to reduce the development or severity of muscle atrophy for improved burn patient outcomes.
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Chiappini, Ciro; Campagnolo, Paola; Almeida, Carina S; Abbassi-Ghadi, Nima; Chow, Lesley W; Hanna, George B; Stevens, Molly M
2015-09-16
Porous silicon nanoneedles can map Cathepsin B activity across normal and tumor human esophageal mucosa. Assembling a peptide-based Cathepsin B cleavable sensor over a large array of nano-needles allows the discrimination of cancer cells from healthy ones in mixed culture. The same sensor applied to tissue can map Cathepsin B activity with high resolution across the tumor margin area of esophageal adenocarcinoma. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Foddai, A C G; Grant, I R
2017-05-01
To validate an optimized peptide-mediated magnetic separation (PMS)-phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk. Inclusivity, specificity and limit of detection 50% (LOD 50 ) of the optimized PMS-phage assay were assessed. Plaques were obtained for all 43 MAP strains tested. Of 12 other Mycobacterium sp. tested, only Mycobacterium bovis BCG produced small numbers of plaques. LOD 50 of the PMS-phage assay was 0·93 MAP cells per 50 ml milk, which was better than both PMS-qPCR and PMS-culture. When individual milks (n = 146) and bulk tank milk (BTM, n = 22) obtained from Johne's affected herds were tested by the PMS-phage assay, viable MAP were detected in 31 (21·2%) of 146 individual milks and 13 (59·1%) of 22 BTM, with MAP numbers detected ranging from 6-948 plaque-forming-units per 50 ml milk. PMS-qPCR and PMS-MGIT culture proved to be less sensitive tests than the PMS-phage assay. The optimized PMS-phage assay is the most sensitive and specific method available for the detection of viable MAP in milk. Further work is needed to streamline the PMS-phage assay, because the assay's multistep format currently makes it unsuitable for adoption by the dairy industry as a screening test. The inclusivity (ability to detect all MAP strains), specificity (ability to detect only MAP) and detection sensitivity (ability to detect low numbers of MAP) of the optimized PMS-phage assay have been comprehensively demonstrated for the first time. © 2017 The Society for Applied Microbiology.
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Mapping membrane activity in undiscovered peptide sequence space using machine learning
Fulan, Benjamin M.; Wong, Gerard C. L.
2016-01-01
There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its “antimicrobialness”) and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide’s minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences. PMID:27849600
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Ruggles, Kelly V; Tang, Zuojian; Wang, Xuya; Grover, Himanshu; Askenazi, Manor; Teubl, Jennifer; Cao, Song; McLellan, Michael D; Clauser, Karl R; Tabb, David L; Mertins, Philipp; Slebos, Robbert; Erdmann-Gilmore, Petra; Li, Shunqiang; Gunawardena, Harsha P; Xie, Ling; Liu, Tao; Zhou, Jian-Ying; Sun, Shisheng; Hoadley, Katherine A; Perou, Charles M; Chen, Xian; Davies, Sherri R; Maher, Christopher A; Kinsinger, Christopher R; Rodland, Karen D; Zhang, Hui; Zhang, Zhen; Ding, Li; Townsend, R Reid; Rodriguez, Henry; Chan, Daniel; Smith, Richard D; Liebler, Daniel C; Carr, Steven A; Payne, Samuel; Ellis, Matthew J; Fenyő, David
2016-03-01
Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song
2002-09-01
Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).
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Cha, S; Leung, P S; Van de Water, J; Tsuneyama, K; Joplin, R E; Ansari, A A; Nakanuma, Y; Schatz, P J; Cwirla, S; Fabris, L E; Neuberger, J M; Gershwin, M E; Coppel, R L
1996-01-01
Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855289
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Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi
2008-08-01
In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.
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Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M
2018-03-28
Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.
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NASA Astrophysics Data System (ADS)
Guo, L. Q.; Hu, Y. W.; Yu, B.; Davis, E.; Irvin, R.; Yan, X. G.; Li, D. Y.
2016-02-01
Titanium dioxide (TiO2) nanotubes are promising for a wide variety of potential applications in energy, biomedical and environmental sectors. However, their low mechanical strength and wide band gap limit their widespread technological use. This article reports our recent efforts to increase the mechanical strength of TiO2 nanotubes with lowered band gap by immobilizing a peptide of D-amino K122-4 (D) onto the nanotubes. Topographies and chemical compositions of the peptide-coated and uncoated TiO2 nanotubular arrays were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy (XPS). Properties of the peptide-coated and uncoated TiO2 nanotubular arrays, including hardness, elastic modulus, electron work function and photocurrent, were evaluated using micromechanical probe, Kelvin Probe and electrochemical system. Effect of the peptide on surface conductivity was also investigated through current mapping and I-V curve analysis with conductive atomic force microscopy. It is demonstrated that the peptide coating simultaneously enhances the mechanical strength, photocatalytic and electrical properties of TiO2 nanotubes.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lien, Shu-Pei; Shih, Yi-Ping; Chen, Hsin-Wei
Three peptides, D1 (amino acid residues 175-201), D2 (a.a. 434-467), and TM (a.a. 1128-1159), corresponding to the spike protein (S) of severe acute respiratory syndrome corona virus (SARS CoV) were synthesized and their immunological functions were investigated in three different animals models (mice, guinea pigs, and rabbits). The peptides mixture formulated either with Freund's adjuvant or synthetic adjuvant Montanide ISA-51/oligodeoxy nucleotide CpG (ISA/CpG) could elicit antisera in immunized animals which were capable of inhibiting SARS/HIV pseudovirus entry into HepG2 cells. The neutralizing epitopes were identified using peptides to block the neutralizing effect of guinea pig antisera. The major neutralizing epitopemore » was located on the D2 peptide, and the amino acid residue was fine mapped to 434-453. In BALB/c mice T-cell proliferation assay revealed that only D2 peptide contained T-cell epitope, the sequence of which corresponded to amino acid residue 434-448. The ISA/CpG formulation generated anti-D2 IgG titer comparable to those obtained from Freund's adjuvant formulation, but generated fewer antibodies against D1 or TM peptides. The highly immunogenic D2 peptide contains both neutralizing and Th cell epitopes. These results suggest that synthetic peptide D2 would be useful as a component of SARS vaccine candidates.« less
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Seyed, Negar; Zahedifard, Farnaz; Safaiyan, Shima; Gholami, Elham; Doustdari, Fatemeh; Azadmanesh, Kayhan; Mirzaei, Maryam; Saeedi Eslami, Nasir; Khadem Sadegh, Akbar; Eslami far, Ali; Sharifi, Iraj; Rafati, Sima
2011-01-01
Background As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis. PMID:21909442
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A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells
NASA Astrophysics Data System (ADS)
Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.
2017-09-01
The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.
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Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg
2016-01-01
Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853
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Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg
2016-07-16
Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.
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Edelmann, Mariola J.
2011-01-01
Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide. PMID:22174558
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Whiteman, Ineka T; Gervasio, Othon L; Cullen, Karen M; Guillemin, Gilles J; Jeong, Erica V; Witting, Paul K; Antao, Shane T; Minamide, Laurie S; Bamburg, James R; Goldsbury, Claire
2009-10-14
In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.
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Smart photonic materials for theranostic applications
NASA Astrophysics Data System (ADS)
Keum, Do Hee; Beack, Songeun; Hahn, Sei Kwang
2017-05-01
We developed melanoidin nanoparticles for in vivo noninvasive photoacoustic mapping of sentinel lymph nodes, photoacoustic tomography of gastro-intestinal tracts, and photothermal ablation cancer therapy. In addition, we developed cell-integrated poly(ethylene glycol) hydrogels for in vivo optogenetic sensing and therapy. Real-time optical readout of encapsulated heat-shock-protein-coupled fluorescent reporter cells made it possible to measure the nanotoxicity of cadmium-based quantum dots in vivo. Using optogenetic cells producing glucagon-like peptide-1, we performed lightcontrolled diabetic therapy for glucose homeostasis. Finally, we developed a smart contact lens composed of biosensors, drug delivery systems, and power sources for the treatment of diabetes as a model disease.
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Niegowska, Magdalena; Delitala, Alessandro; Pes, Giovanni Mario; Delitala, Giuseppe
2017-01-01
Latent Autoimmune Diabetes in Adults (LADA) is a slowly progressing form of immune-mediated diabetes that combines phenotypical features of type 2 diabetes (T2D) with the presence of islet cell antigens detected in type 1 diabetes (T1D). Heterogeneous clinical picture have led to the classification of patients based on the levels of antibodies against glutamic acid decarboxylase 65 (GADA) that correlate with clinical phenotypes closer to T1D or T2D when GADA titers are high or low, respectively. To date, LADA etiology remains elusive despite numerous studies investigating on genetic predisposition and environmental risk factors. To our knowledge, this is the first study aimed at evaluation of a putative role played by Mycobacterium avium subsp. paratuberculosis (MAP) as an infective agent in LADA pathogenesis. MAP is known to cause chronic enteritis in ruminants and has been associated with autoimmune disorders in humans. We analyzed seroreactivity of 223 Sardinian LADA subjects and 182 healthy volunteers against MAP-derived peptides and their human homologs of proinsulin and zinc transporter 8 protein. A significantly elevated positivity for MAP/proinsulin was detected among patients, with the highest prevalence in the 32-41-year-old T1D-like LADA subgroup, supporting our hypothesis of a possible MAP contribution in the development of autoimmunity. PMID:28472070
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Norcross, Michael A.; Luo, Shen; Lu, Li; Boyne, Michael T.; Gomarteli, Mary; Rennels, Aaron D.; Woodcock, Janet; Margulies, David H.; McMurtrey, Curtis; Vernon, Stephen; Hildebrand, William H.; Buchli, Rico
2014-01-01
Background Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B*57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B*57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B*57:01 from abacavir-treated cells. Design and methods An HLA-B*57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B*57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B*57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B*57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B*57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B*57:01 with high affinity that was not altered by abacavir addition. Conclusion Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B*57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity. PMID:22617051
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Norcross, Michael A; Luo, Shen; Lu, Li; Boyne, Michael T; Gomarteli, Mary; Rennels, Aaron D; Woodcock, Janet; Margulies, David H; McMurtrey, Curtis; Vernon, Stephen; Hildebrand, William H; Buchli, Rico
2012-07-17
Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B57:01 from abacavir-treated cells. An HLA-B57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B57:01 with high affinity that was not altered by abacavir addition. Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.
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GuiTope: an application for mapping random-sequence peptides to protein sequences.
Halperin, Rebecca F; Stafford, Phillip; Emery, Jack S; Navalkar, Krupa Arun; Johnston, Stephen Albert
2012-01-03
Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.
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NASA Astrophysics Data System (ADS)
Switzar, Linda; Nicolardi, Simone; Rutten, Julie W.; Oberstein, Saskia A. J. Lesnik; Aartsma-Rus, Annemieke; van der Burgt, Yuri E. M.
2016-01-01
Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S-S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the "parent" disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.
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Ferro, Myriam; Tardif, Marianne; Reguer, Erwan; Cahuzac, Romain; Bruley, Christophe; Vermat, Thierry; Nugues, Estelle; Vigouroux, Marielle; Vandenbrouck, Yves; Garin, Jérôme; Viari, Alain
2008-05-01
PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.
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NASA Astrophysics Data System (ADS)
Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang
2013-03-01
A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.
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Rose, M C; Kaufman, B; Martin, B M
1989-05-15
Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.
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Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H
2000-04-01
VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.
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Flynn, J N; Cannon, C A; Neil, J C; Jarrett, O
1997-01-01
Cats were immunized with a 46-residue multiepitopic synthetic peptide of feline immunodeficiency virus (FIV) comprising immunodominant epitopes present in the third variable domain of the envelope glycoprotein, transmembrane glycoprotein (TM), and p24 Gag core protein, using Quil A as an adjuvant. All vaccinated cats developed a humoral response which recognized the synthetic peptide immunogen and the intact viral core and envelope proteins. A FIV Gag- and Env-specific effector cytotoxic T-lymphocyte response was also detected in the peripheral blood of vaccinated cats, which peaked at week 30. This response appeared to be major histocompatibility complex restricted. Epitope mapping studies revealed that both the cellular and humoral immune responses were directed principally to a peptide within the TM glycoprotein, CNQNQFFCK. However, vaccination did not confer protection when cats were challenged with the Petaluma isolate of FIV at week 35. PMID:9311839
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The Extended Granin Family: Structure, Function, and Biomedical Implications
Bartolomucci, Alessandro; Possenti, Roberta; Mahata, Sushil K.; Fischer-Colbrie, Reiner; Loh, Y. Peng
2011-01-01
The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers. PMID:21862681
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The extended granin family: structure, function, and biomedical implications.
Bartolomucci, Alessandro; Possenti, Roberta; Mahata, Sushil K; Fischer-Colbrie, Reiner; Loh, Y Peng; Salton, Stephen R J
2011-12-01
The chromogranins (chromogranin A and chromogranin B), secretogranins (secretogranin II and secretogranin III), and additional related proteins (7B2, NESP55, proSAAS, and VGF) that together comprise the granin family subserve essential roles in the regulated secretory pathway that is responsible for controlled delivery of peptides, hormones, neurotransmitters, and growth factors. Here we review the structure and function of granins and granin-derived peptides and expansive new genetic evidence, including recent single-nucleotide polymorphism mapping, genomic sequence comparisons, and analysis of transgenic and knockout mice, which together support an important and evolutionarily conserved role for these proteins in large dense-core vesicle biogenesis and regulated secretion. Recent data further indicate that their processed peptides function prominently in metabolic and glucose homeostasis, emotional behavior, pain pathways, and blood pressure modulation, suggesting future utility of granins and granin-derived peptides as novel disease biomarkers. Copyright © 2011 by The Endocrine Society
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Vasicek, Lisa; O'Brien, John P.; Browning, Karen S.; Tao, Zhihua; Liu, Hung-Wen; Brodbelt, Jennifer S.
2012-01-01
A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C. PMID:22393264
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Atassi, M Zouhair; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger; Dolimbek, Behzod Z
2012-01-01
We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs. Copyright © 2011. Published by Elsevier GmbH.
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Su, Shih-Ping; McArthur, Jason D; Andrew Aquilina, J
2010-07-01
Low molecular weight (LMW) peptides, derived from the breakdown of the major eye lens proteins, the crystallins, accumulate in the human lens with age. These LMW peptides are associated with age-related lens opacity and cataract, with some shown to inhibit the chaperone activity of alpha-crystallin. However, the mechanism(s) giving rise to the production of these peptides, as well as their distribution within the lens, are not well understood. In this study, we have mapped the distribution of these crystallin-derived peptides present in human lenses of different ages using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Our data showed that most of these LMW peptides emerge in the lens at early middle-age, with peptides greater than 1778 Da in mass being confined to the water insoluble fractions, and to a lesser extent the water soluble fractions of older lenses. MALDI-IMS analyses showed that four peptides, derived from alphaA-, alphaB- and gammaS-crystallins, were confined to the lens nuclear fibre cells upon emergence during early middle-age, but were present in both the cortex and nucleus of old lenses. In contrast, another major peptide, derived from the C-terminal breakdown of betaA3-crystallin, was present in the cortical and nuclear regions of both young and old lenses. A comparison between age-matched cataractous and non-cataractous lenses showed no distinct differences in LMW peptide profiles, indicating that although cataract may be a potential consequence caused by the emergence of these peptides, it does not contribute directly to the peptide-generating process. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.
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Reddy, G; Nanduri, V B; Basu, A; Modak, M J
1991-08-20
Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.
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Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka
NASA Astrophysics Data System (ADS)
Nielsen-Marsh, Christina M.; Ostrom, Peggy H.; Gandhi, Hasand; Shapiro, Beth; Cooper, Alan; Hauschka, Peter V.; Collins, Matthew J.
2002-12-01
We report the first complete sequences of the protein osteocalcin from small amounts (20 mg) of two bison bone (Bison priscus) dated to older than 55.6 ka and older than 58.9 ka. Osteocalcin was purified using new gravity columns (never exposed to protein) followed by microbore reversed-phase high-performance liquid chromatography. Sequencing of osteocalcin employed two methods of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS): peptide mass mapping (PMM) and post-source decay (PSD). The PMM shows that ancient and modern bison osteocalcin have the same mass to charge (m/z) distribution, indicating an identical protein sequence and absence of diagenetic products. This was confirmed by PSD of the m/z 2066 tryptic peptide (residues 1 19); the mass spectra from ancient and modern peptides were identical. The 129 mass unit difference in the molecular ion between cow (Bos taurus) and bison is caused by a single amino-acid substitution between the taxa (Trp in cow is replaced by Gly in bison at residue 5). Bison mitochondrial control region DNA sequences were obtained from the older than 55.6 ka fossil. These results suggest that DNA and protein sequences can be used to directly investigate molecular phylogenies over a considerable time period, the absolute limit of which is yet to be determined.
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NASA Astrophysics Data System (ADS)
Thibault, P.; Pleasance, S.; Laycock, M. V.; Mackay, R. M.; Boyd, R. K.
1991-12-01
An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography--mass spectrometry (LC--MS) and LC--MS--MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC--MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC--MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray® LC--MS interface (pneumatically assisted electrospray).
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Stable isotope, site-specific mass tagging for protein identification
Chen, Xian
2006-10-24
Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.
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A Crosslinker Based on a Tethered Electrophile for Mapping Kinase-Substrate Networks
Riel-Mehan, Megan M; Shokat, Kevan M
2014-01-01
SUMMARY Despite the continuing progress made towards mapping kinase signaling networks, there are still many phosphorylation events for which the responsible kinase has not yet been identified. We are interested in addressing this problem through forming covalent crosslinks between a peptide substrate and the corresponding phosphorylating kinase. Previously we reported a dialdehyde-based kinase binding probe capable of such a reaction with a peptide containing a cysteine substituted for the phosphorylatable ser/thr/tyr residue. Here, we examine the yield of a previously reported dialdehyde-based probe, and report that the dialdehyde based probes possesses a significant limitation in terms of crosslinked kinase-substrate product yield. To address this limitation, we develop a crosslinking scheme based on a kinase activity-based probe, and this new cross-linker provides an increase in efficiency and substrate specificity, including in the context of cell lysate. PMID:24746561
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Honda, Eduardo R; Zanchi, Fernando; Rios, Katiuscia; Lira, Ednaldo; DeusileneVieira; da Silva, Luiz H P; De Paula, Sergio O
2012-12-01
Viruses belonging to the Flaviviridae family are found and distributed in most of the tropical and sub-tropical regions of the world. The genus has more than 56 members, most of which cause clinical symptoms in humans. The clinical diagnosis of dengue requires laboratory confirmation because of the similarity of symptoms with a series of other acute fevers and the primary use antibodies or antigens for detection. In this work, peptides E(1) and E(2) of the envelope protein (E) of the dengue virus were mapped using bioinformatics methods. These peptides were then expressed in a prokaryotic system and purified. An indirect ELISA for antibodies IgG and IgM from laboratory samples previously characterised was then used with the peptides to detect anti-dengue antibodies. For IgG using the peptide E(1), the sensitivity of the indirect ELISA was 88.3% and the specificity was 56%; using the peptide E(2), the sensitivity was 90% and the specificity was 59%; and using a combination of both peptides, the sensitivity was 93.3% and the specificity was 78%. For IgM using the peptide E(1), the sensitivity was 88% and the specificity was 66%; using the peptide E(2), the sensitivity was 88% and the specificity was 69%; and when used in combination, the peptides E(1)/E(2) demonstrated a sensitivity of 90% and a specificity of 86%. These results indicate that the use of the E(1) and E(2) peptides of the E protein are an alternative for serological diagnosis of dengue fever. Copyright © 2012 Elsevier B.V. All rights reserved.
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Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin.
Yan, Yuetian; Chen, Guodong; Wei, Hui; Huang, Richard Y-C; Mo, Jingjie; Rempel, Don L; Tymiak, Adrienne A; Gross, Michael L
2014-12-01
Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin ((10)Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.
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Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin
NASA Astrophysics Data System (ADS)
Yan, Yuetian; Chen, Guodong; Wei, Hui; Huang, Richard Y.-C.; Mo, Jingjie; Rempel, Don L.; Tymiak, Adrienne A.; Gross, Michael L.
2014-12-01
Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.
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Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies.
Siakkou, H; Jahn, S; Kienzle, N; Ulrich, R; Grötzinger, C; Schneider, T; Kohleisen, B; Pauli, G; Spohn, R; Jung, G
1993-01-01
A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83-93, 175-190 and 86-166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.
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Grosvenor, Anita J; Haigh, Brendan J; Dyer, Jolon M
2014-11-01
The extent to which nutritional and functional benefit is derived from proteins in food is related to its breakdown and digestion in the body after consumption. Further, detailed information about food protein truncation during digestion is critical to understanding and optimising the availability of bioactives, in controlling and limiting allergen release, and in minimising or monitoring the effects of processing and food preparation. However, tracking the complex array of products formed during the digestion of proteins is not easily accomplished using classical proteomics. We here present and develop a novel proteomic approach using isobaric labelling to mapping and tracking protein truncation and peptide release during simulated gastric digestion, using bovine lactoferrin as a model food protein. The relative abundance of related peptides was tracked throughout a digestion time course, and the effect of pasteurisation on peptide release assessed. The new approach to food digestion proteomics developed here therefore appears to be highly suitable not only for tracking the truncation and relative abundance of released peptides during gastric digestion, but also for determining the effects of protein modification on digestibility and potential bioavailability.
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The FMRFamide-Like Peptide Family in Nematodes
Peymen, Katleen; Watteyne, Jan; Frooninckx, Lotte; Schoofs, Liliane; Beets, Isabel
2014-01-01
In the three decades since the FMRFamide peptide was isolated from the mollusk Macrocallista nimbosa, structurally similar peptides sharing a C-terminal RFamide motif have been identified across the animal kingdom. FMRFamide-like peptides (FLPs) represent the largest known family of neuropeptides in invertebrates. In the phylum Nematoda, at least 32 flp-genes are classified, making the FLP system of nematodes unusually complex. The diversity of the nematode FLP complement is most extensively mapped in Caenorhabditis elegans, where over 70 FLPs have been predicted. FLPs have shown to be expressed in the majority of the 302 C. elegans neurons including interneurons, sensory neurons, and motor neurons. The vast expression of FLPs is reflected in the broad functional repertoire of nematode FLP signaling, including neuroendocrine and neuromodulatory effects on locomotory activity, reproduction, feeding, and behavior. In contrast to the many identified nematode FLPs, only few peptides have been assigned a receptor and there is the need to clarify the pathway components and working mechanisms of the FLP signaling network. Here, we review the diversity, distribution, and functions of FLPs in nematodes. PMID:24982652
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A 31-residue peptide induces aggregation of tau’s microtubule-binding region in cells
Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Achyraya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J.S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; DeGrado, William F.
2018-01-01
The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer’s disease. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behavior of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show here that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fiber diffraction, hydrogen-deuterium exchange and solids NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide. PMID:28837163
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Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J
2011-03-01
Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.
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SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.
Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P
2016-07-01
The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*
Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.
2016-01-01
The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445
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Baum, Florian; Fedorova, Maria; Ebner, Jennifer; Hoffmann, Ralf; Pischetsrieder, Monika
2013-12-06
Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from α(S1)-casein, followed by β-casein, and α(S2)-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nielson, Carrie M.; Wiedrick, Jack; Shen, Jian
Accelerated bone loss significantly increases the risk of osteoporosis and fracture. The mechanisms underlying bone loss remain incompletely understood, and there are few available biomarkers. We utilized a novel proteomics approach to identify serum peptides and proteins associated with bone loss in 1967 older men who were randomly chosen from the Osteoporotic Fracture in Men Study (MrOS study) (age ≥ 65 yrs). Men had 2-3 measures of femoral neck BMD over an average follow-up of 4.6 years. Change in BMD was estimated and then categorized into three groups: maintained BMD (n=453), expected loss (n=1185) and accelerated loss (n=237). A liquidmore » chromatography–ion mobility separation-mass spectrometry (LC-IMS-MS) proteomics platform was used to identify and quantify peptides from serum proteins. The whole cohort was randomly divided into discovery (N= 960) and validation (N= 915) sub-cohorts. Linear regression models and a random forest approach were used to discover differentially abundant individual peptides and a proteomic signature that distinguished individuals with accelerated bone loss from those who maintained BMD. Network analyses were performed using the MetaCore knowledgebase. We identified 12 peptides that were associated with BMD loss in both discovery (P< 0.1 FDR) and replication sub-cohorts (P<0.05). Those 12 peptides mapped to the following proteins: ALS, LYVE1, RNAS1, C2, ICOSL, C163A, C7, HEMO, CD14, CERU, CRAC1 and CD59. Meta-analysis of peptidesassociated with bone loss identified 6 additional proteins including GRP78, IGF-2, SHBG, ENPP2, IBP2 and IBP6. We also identified a proteomic signature that was predictive of BMD loss with a discriminative value similar to serum bone marker carboxy-terminal collagen crosslink peptide (CTX). Interestingly, combining the proteomic signature with CTX significantly improved the ability to discriminate men with accelerated loss. In summary, we have identified potential new biomarkers for bone loss that provide a more in depth understanding of its pathophysiology.« less
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MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes
Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V.; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J.; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wiśniewski, Jacek R.; Jun, Wang; Mann, Matthias
2007-01-01
Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools. PMID:17090601
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A surface plasmon resonance assay for characterisation and epitope mapping of anti-GLP-1 antibodies.
Thomsen, Lasse; Gurevich, Leonid
2018-04-19
The incretin hormone glucagon-like peptide-1 (GLP-1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP-1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP-1 receptor agonists. Surface plasmon resonance (SPR) facilitates real-time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme-linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti-GLP-1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti-GLP-1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10 3 to 4.54 × 10 3 M -1 s -1 and dissociation rates of 3.56 × 10 -5 to 1.56 × 10 -3 s -1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC p < 0). Pair-wise epitope mapping was performed on captured anti-GLP-1 antibodies followed by subsequent interaction with GLP-1 (7-36) and other anti-GLP-1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti-GLP-1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool. Copyright © 2018 John Wiley & Sons, Ltd.
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Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin
2015-10-14
The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.
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Riffle, Michael; Merrihew, Gennifer E; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N; Noble, William S; MacCoss, Michael J
2015-11-01
Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/ . Graphical Abstract ᅟ.
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Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D; Nesvizhskii, Alexey I; Sung, Ting-Yi; Chen, Yu-Ju
2015-09-04
Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.
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Carré-Eusèbe, D; Lederer, F; Lê, K H; Elsevier, S M
1991-01-01
Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid. Images Fig. 1. Fig. 3. Fig. 4. PMID:1854346
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Wang, Jing J; Al Kindi, Mahmood A; Colella, Alex D; Dykes, Lukah; Jackson, Michael W; Chataway, Tim K; Reed, Joanne H; Gordon, Tom P
2016-12-01
We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity. Copyright © 2016 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.
2015-11-01
Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.
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Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica
2013-04-01
The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya
Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic andmore » germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.« less
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Saito, Mak A; Dorsk, Alexander; Post, Anton F; McIlvin, Matthew R; Rappé, Michael S; DiTullio, Giacomo R; Moran, Dawn M
2015-10-01
Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8 ± 1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13 ± 15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ∼4 × 10(7) , 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.
Nury, Catherine; Redeker, Virginie; Dautrey, Sébastien; Romieu, Anthony; van der Rest, Guillaume; Renard, Pierre-Yves; Melki, Ronald; Chamot-Rooke, Julia
2015-02-03
The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.
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Griaud, François; Denefeld, Blandine; Lang, Manuel; Hensinger, Héloïse; Haberl, Peter; Berg, Matthias
2017-07-01
Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.
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Hill, Ryan C.; Wither, Matthew J.; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C.
2015-01-01
Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757
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Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.
Kunys, Andrew R; Lian, Wenlong; Pei, Dehua
2012-12-01
One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.
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Hepatitis C Virus Antigenic Convergence
Campo, David S.; Dimitrova, Zoya; Yokosawa, Jonny; Hoang, Duc; Perez, Nestor O.; Ramachandran, Sumathi; Khudyakov, Yury
2012-01-01
Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development. PMID:22355779
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Human gastrin-releasing peptide gene is located on chromosome 18.
Naylor, S L; Sakaguchi, A Y; Spindel, E; Chin, W W
1987-01-01
Gastrin-releasing peptide (GRP), a bombesin-like peptide, increases plasma levels of gastrin, pancreatic polypeptide, glucagon, gastric inhibitory peptide, and insulin. GRP is produced in large quantities by small-cell lung cancer and acts as a growth factor for these cells. To determine if chromosomal changes in small-cell lung cancer are related to the expression of GRP, we chromosomally mapped the gene using human-mouse somatic cell hybrids. Twenty hybrids, characterized for human chromosomes, were analyzed by Southern filter hybridization of DNA digested with EcoRI. Human DNA cut with EcoRI yields a major band of 6.8 kb and a minor band of 11.3 kb. The 6.8 kb band segregated concordantly with chromosome 18 and the marker peptidase A. The chromosome 3 abnormalities seen in small-cell lung cancer do not correlate with the chromosomal location of GRP, suggesting that the elevated expression of this gene may be due to mechanisms other than chromosomal rearrangement.
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Tipu, Hamid Nawaz
2016-02-01
To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.
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Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica
2017-09-01
Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.
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The Pig PeptideAtlas: A resource for systems biology in animal production and biomedicine.
Hesselager, Marianne O; Codrea, Marius C; Sun, Zhi; Deutsch, Eric W; Bennike, Tue B; Stensballe, Allan; Bundgaard, Louise; Moritz, Robert L; Bendixen, Emøke
2016-02-01
Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system-wide observations, and cross-species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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The Pig PeptideAtlas: a resource for systems biology in animal production and biomedicine
Hesselager, Marianne O.; Codrea, Marius C.; Sun, Zhi; Deutsch, Eric W.; Bennike, Tue B.; Stensballe, Allan; Bundgaard, Louise; Moritz, Robert L.; Bendixen, Emøke
2016-01-01
Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system wide observations, and cross species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this manuscript demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research. PMID:26699206
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Kurz, Thorsten; Hoffjan, Sabine; Hayes, M Geoffrey; Schneider, Dan; Nicolae, Raluca; Heinzmann, Andrea; Jerkic, Sylvija P; Parry, Rod; Cox, Nancy J; Deichmann, Klaus A; Ober, Carole
2006-08-01
Genome-wide linkage scans to identify asthma susceptibility loci have revealed many linked regions, including a broad region on chromosome 5p. To identify a 5p-linked asthma or bronchial hyperresponsiveness (BHR) locus. We performed fine mapping and positional candidate studies of this region in the Hutterites and an outbred case-control sample from Germany by genotyping 89 single nucleotide polymorphisms (SNPs) in 22 genes. SNP and haplotype analyses were performed. Three genes in a distal region (zinc finger RNA binding protein [ZFR], natriuretic peptide receptor C, and a disintegrin and metalloproteinase domain with thrombospondin type 1 motif [ADAMTS12]) were associated with BHR, whereas 4 genes in a proximal region (prolactin receptor, IL-7 receptor [IL7R], leukemia inhibitory factor receptor [LIFR], and prostaglandin E4 receptor [PTGER4]) were associated with asthma symptoms in the Hutterites. Furthermore, nearly the entire original linkage signal in the Hutterites was generated by individuals who had the risk-associated alleles in ZFR3, natriuretic peptide receptor C, ADAMTS12, LIFR, and PTGER4. Variation in ADAMTS12, IL7R, and PTGER4 were also associated with asthma in the outbred Germans, and the frequencies of long-range haplotypes composed of SNPs at ZFR, ADAMTS12, IL7R, LIFR, and PTGER4 were significantly different between both the German and Hutterite cases and controls. There is little linkage disequilbrium between alleles in these 2 regions in either population. These results suggest that a broad region on 5p, separated by >9 Mb, harbors at least 2 and possibly 5 asthma or BHR susceptibility loci. These findings are consistent with the hypothesis that regions providing evidence for linkage in multiple populations may, in fact, house more than 1 susceptibility locus, as appears to be the case for the linked region on 5p. Identifying asthma or BHR genes could lead to novel therapeutic approaches.
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2016-10-01
identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to identify novel PCSC-targeting chemicals. In the past...display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to...Goals of the Project (SOW): Aim 1: To perform phage display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides
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Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.
Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H
2015-10-01
To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.
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Ma, Ji-Hong; Yang, Fu-Ru; Yu, Hai; Zhou, Yan-Jun; Li, Guo-Xin; Huang, Meng; Wen, Feng; Tong, Guangzhi
2013-07-09
Vaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses. Our results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds' adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus. Based on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.
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Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk.
Chen, Y F; Zhao, W J; Wu, R N; Sun, Z H; Zhang, W Y; Wang, J C; Bilige, M; Zhang, H P
2014-12-01
Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Urotensin II in Invertebrates: From Structure to Function in Aplysia californica
Romanova, Elena V.; Sasaki, Kosei; Alexeeva, Vera; Vilim, Ferdinand S.; Jing, Jian; Richmond, Timothy A.; Weiss, Klaudiusz R.; Sweedler, Jonathan V.
2012-01-01
Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan. PMID:23144960
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Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen
2016-07-01
Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.
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Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization
2012-01-01
Background To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents. PMID:22264266
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O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.
Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M
2018-05-05
The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.
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Däumer, Martin P; Schneider, Beate; Giesen, Doris M; Aziz, Sheriff; Kaiser, Rolf; Kupfer, Bernd; Schneweis, Karl E; Schneider-Mergener, Jens; Reineke, Ulrich; Matz, Bertfried; Eis-Hübinger, Anna M
2011-05-01
Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.
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Tu, Maolin; Liu, Hanxiong; Zhang, Ruyi; Chen, Hui; Mao, Fengjiao; Cheng, Shuzhen; Lu, Weihong; Du, Ming
2018-04-25
Casein hydrolysates exert various biological activities, and the responsible functional peptides are being identified from them continuously. In this study, the tryptic casein hydrolysate was fractionated by an ultrafiltration membrane (3 kDa), and the peptides were identified by capillary electrophoresis-quadrupole-time-of-flight-tandem mass spectrometry. Meanwhile, in silico methods were used to analyze the toxicity, solubility, stability, and affinity between the peptides and angiotensin-I-converting enzyme (ACE). Finally, a new angiotensin-I-converting enzyme inhibitory (ACEI) peptide, EKVNELSK, derived from α s1 -casein (fragment 35-42) was screened. The half maximal inhibitory concentration value of the peptide is 5.998 mM, which was determined by a high-performance liquid chromatography method. The Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform molecular docking to propose the potential mechanisms underlying the ACEI activity of the peptide. These results indicated that EKVNELSK is a new ACEI peptide identified from casein hydrolysate.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhu, Yuanyuan; Munro, Catherine J.; Olszta, Matthew J.
In this work, we showcase that through precise control of the electron dose rate, state-of-the-art large solid angle energy dispersive X-ray spectroscopy (EDS) mapping in aberration-corrected scanning transmission electron microscope (STEM) is capable of faithful and unambiguous chemical characterization of the Pt and Pd distribution in a peptide-mediated nanosystem. This low-dose-rate recording scheme adds another dimension of flexibility to the design of elemental mapping experiments, and holds significant potential for extending its application to a wide variety of beam sensitive hybrid nanostructures.
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2016-09-01
Inhibition of MAP kinase pathway prevents plasma protrusions Next we used a selective inhibitor of MAP kinases , PD98059, to address whether we can...from human patients harbor AKT1 and that AKT1 kinase activity is sustained in these particles, nominating them as active signaling platforms...with the extracellular matrix (ECM) and extracellular molecules (2). Though many classic extracellular signaling molecules (e.g., hormones, peptide
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Wang, Yufei; Whittall, Trevor; McGowan, Edward; Younson, Justine; Kelly, Charles; Bergmeier, Lesley A; Singh, Mahavir; Lehner, Thomas
2005-03-15
The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.
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Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena
2012-10-01
Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.
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Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun
2017-01-30
Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches. We hypothesize that more quantifiable spectra and peptides in a protein, even including less confident peptides, could help reduce variations and improve protein quantification. Hence the peptide retrieval strategy was developed and evaluated in two spike-in sample sets with different LC-MS/MS variations using both MS1- and MS2-based quantitative approach. The list of confidently identified proteins using the standard target-decoy search strategy was fixed and more spectra/peptides with less confidence matched to confident proteins were retrieved. However, the total peptide-spectrum-match false discovery rate (PSM FDR) after retrieval analysis was still controlled at a confident level of FDR≤1%. As expected, the penalty for occasionally incorporating incorrect peptide identifications is negligible by comparison with the improvements in quantitative performance. More quantifiable peptides, lower missing value rate, better quantitative accuracy and precision were significantly achieved for the same protein identifications by this simple strategy. This strategy is theoretically applicable for any quantitative approaches in proteomics and thereby provides more quantitative information, especially on low-abundance proteins. Published by Elsevier B.V.
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The rate of cis-trans conformation errors is increasing in low-resolution crystal structures.
Croll, Tristan Ian
2015-03-01
Cis-peptide bonds (with the exception of X-Pro) are exceedingly rare in native protein structures, yet a check for these is not currently included in the standard workflow for some common crystallography packages nor in the automated quality checks that are applied during submission to the Protein Data Bank. This appears to be leading to a growing rate of inclusion of spurious cis-peptide bonds in low-resolution structures both in absolute terms and as a fraction of solved residues. Most concerningly, it is possible for structures to contain very large numbers (>1%) of spurious cis-peptide bonds while still achieving excellent quality reports from MolProbity, leading to concerns that ignoring such errors is allowing software to overfit maps without producing telltale errors in, for example, the Ramachandran plot.
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Castro, K L; Duarte, C G; Ramos, H R; Machado de Avila, R A; Schneider, F S; Oliveira, D; Freitas, C F; Kalapothakis, E; Ho, P L; Chávez-Olortegui, C
2015-01-01
The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Schwenk, Jochen M; Omenn, Gilbert S; Sun, Zhi; Campbell, David S; Baker, Mark S; Overall, Christopher M; Aebersold, Ruedi; Moritz, Robert L; Deutsch, Eric W
2017-12-01
Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization's Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ∼43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely mapping peptides of nine amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ∼2000 proteins. Finally, we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.
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Shao, Wenguang; Pedrioli, Patrick G A; Wolski, Witold; Scurtescu, Cristian; Schmid, Emanuel; Courcelles, Mathieu; Schuster, Heiko; Kowalewski, Daniel; Marino, Fabio; Arlehamn, Cecilia S L; Vaughan, Kerrie; Peters, Bjoern; Sette, Alessandro; Ottenhoff, Tom H M; Meijgaarden, Krista E; Nieuwenhuizen, Natalie; Kaufmann, Stefan H E; Schlapbach, Ralph; Castle, John C; Nesvizhskii, Alexey I; Nielsen, Morten; Deutsch, Eric W; Campbell, David S; Moritz, Robert L; Zubarev, Roman A; Ytterberg, Anders Jimmy; Purcell, Anthony W; Marcilla, Miguel; Paradela, Alberto; Wang, Qi; Costello, Catherine E; Ternette, Nicola; van Veelen, Peter A; van Els, Cécile A C M; de Souza, Gustavo A; Sollid, Ludvig M; Admon, Arie; Stevanovic, Stefan; Rammensee, Hans-Georg; Thibault, Pierre; Perreault, Claude; Bassani-Sternberg, Michal
2018-01-01
Abstract Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts. PMID:28985418
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Basu, A; Williams, K R; Modak, M J
1987-07-15
Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.
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Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry
Mendoza, Vanessa Leah; Vachet, Richard W.
2009-01-01
For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300
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Development of Bacterial Display Peptides for use in Biosensing Applications
2012-09-01
performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB...reagent, affinity reagent, bacterial display, multi-scale modeling, docking, protective antigen , SEB, biosensing 16. SECURITY CLASSIFICATION OF: 17...performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be
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DNA β-Amyloid1–42 Trimer Immunization for Alzheimer Disease in a Wild-Type Mouse Model
Lambracht-Washington, Doris; Qu, Bao-Xi; Fu, Min; Eagar, Todd N.; Stüve, Olaf; Rosenberg, Roger N.
2010-01-01
Context DNA β-amyloid1–42 (Aβ42) trimer immunization was developed to produce specific T helper 2 cell (TH2)–type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Aβ42 peptide that occur in the brain of patients with AD. Objective To compare the immune response in wild-type mice after immunization with DNA Aβ42 trimer and Aβ42 peptide. Design and Intervention Wild-type mice received either 4 µg of DNA Aβ42 trimer immunization administered with gene gun (n=8) or intraperitoneal injection of 100 µg of human Aβ42 peptide with the adjuvant Quil A (n=8). Titers, epitope mapping, and isotypes of the Aβ42-specific antibodies were analyzed. Main Outcome Measures Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Aβ42-specific antibodies, and T-cell activation. Results DNA Aβ42 trimer immunization resulted in antibody titers with a mean of 15 µg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a TH2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed TH1/TH2 immune response (IgG1/IgG2a ratio of 1) (P<.001). No increased T-cell proliferation was observed in the DNA-immunized mice (P=.03). Conclusion In this preliminary study in a wild-type mouse model, DNA Aβ42 trimer immunization protocol produced a TH2 immune response and appeared to have low potential to cause an inflammatory T-cell response. PMID:19861672
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DNA beta-amyloid(1-42) trimer immunization for Alzheimer disease in a wild-type mouse model.
Lambracht-Washington, Doris; Qu, Bao-Xi; Fu, Min; Eagar, Todd N; Stüve, Olaf; Rosenberg, Roger N
2009-10-28
DNA beta-amyloid(1-42) (Abeta42) trimer immunization was developed to produce specific T helper 2 cell (T(H)2)-type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Abeta42 peptide that occur in the brain of patients with AD. To compare the immune response in wild-type mice after immunization with DNA Abeta42 trimer and Abeta42 peptide. Wild-type mice received either 4 microg of DNA Abeta42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 microg of human Abeta42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Abeta42-specific antibodies were analyzed. Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Abeta42-specific antibodies, and T-cell activation. DNA Abeta42 trimer immunization resulted in antibody titers with a mean of 15 microg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T(H)2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T(H)1/T(H)2 immune response (IgG1/IgG2a ratio of 1) (P < .001). No increased T-cell proliferation was observed in the DNA-immunized mice (P = .03). In this preliminary study in a wild-type mouse model, DNA Abeta42 trimer immunization protocol produced a T(H)2 immune response and appeared to have low potential to cause an inflammatory T-cell response.
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Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R
2001-03-15
Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.
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Metabolic flux analysis using 13C peptide label measurements
USDA-ARS?s Scientific Manuscript database
13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...
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Granholm, Ann-Charlotte; Bimonte-Nelson, Heather A.; Moore, Alfred B.; Nelson, Matthew E.; Freeman, Linnea R.; Sambamurti, Kumar
2009-01-01
Diets rich in cholesterol and/or saturated fats have been shown to be detrimental to cognitive performance. Therefore, we fed a cholesterol (2%) and saturated fat (hydrogenated coconut oil, Sat Fat 10%) diet to 16-month old rats for 8 weeks to explore the effects on the working memory performance of middle-aged rats. Lipid profiles revealed elevated plasma triglycerides, total cholesterol, HDL, and LDL for the Sat-Fat group as compared to an iso-caloric control diet (12% soybean oil). Weight gain and food consumption were similar in both groups. Sat-Fat treated rats committed more working memory errors in the water radial arm maze, especially at higher memory loads. Cholesterol, amyloid-β peptide of 40 (Aβ40) or 42 (Aβ42) residues, and nerve growth factor in cortical regions was unaffected, but hippocampal Map-2 staining was reduced in rats fed a Sat-Fat diet, indicating a loss of dendritic integrity. Map-2 reduction correlated with memory errors. Microglial activation, indicating inflammation and/or gliosis, was also observed in the hippocampus of Sat-Fat fed rats. These data suggest that saturated fat, hydrogenated fat and cholesterol can profoundly impair memory and hippocampal morphology. PMID:18560126
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Transport Functions Dominate the SAR11 Metaproteome at Low-Nutrient Extremes in the Sargasso Sea
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sowell, Sarah M.; Wilhelm, Larry; Norbeck, Angela D.
2009-01-01
The northwestern Sargasso Sea is part of the North Atlantic subtropical oceanic gyre that is characterized as seasonally oligotrophic with pronounced stratification in the summer and autumn. Essentially a marine desert, the biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorous and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary LC-tandem mass spectrometry to study the composition of microbial proteomes in surface samples collected in September 2005. A total of 2279 peptides that mapped to 236 SAR11 proteins, andmore » 3208 peptides that mapped to 404 Synechococcus proteins, were detected. Mass spectra from SAR11 periplasmic binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars, and spermidine. Although the data showed that a large fraction of microbial protein synthesis in the Sargasso Sea is devoted to inorganic and organic nutrient acquisition, the proteomes of both SAR11 and Synechococcus also indicated that these populations were actively growing. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme but sufficient to sustain microbial community activity.« less
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Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju
2016-01-01
Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522
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Evolution of a mass spectrometry-grade protease with PTM-directed specificity.
Tran, Duc T; Cavett, Valerie J; Dang, Vuong Q; Torres, Héctor L; Paegel, Brian M
2016-12-20
Mapping posttranslational modifications (PTMs), which diversely modulate biological functions, represents a significant analytical challenge. The centerpiece technology for PTM site identification, mass spectrometry (MS), requires proteolytic cleavage in the vicinity of a PTM to yield peptides for sequencing. This requirement catalyzed our efforts to evolve MS-grade mutant PTM-directed proteases. Citrulline, a PTM implicated in epigenetic and immunological function, made an ideal first target, because citrullination eliminates arginyl tryptic sites. Bead-displayed trypsin mutant genes were translated in droplets, the mutant proteases were challenged to cleave bead-bound fluorogenic probes of citrulline-dependent proteolysis, and the resultant beads (1.3 million) were screened. The most promising mutant efficiently catalyzed citrulline-dependent peptide bond cleavage (k cat /K M = 6.9 × 10 5 M -1 ⋅s -1 ). The resulting C-terminally citrullinated peptides generated characteristic isotopic patterns in MALDI-TOF MS, and both a fragmentation product y 1 ion corresponding to citrulline (176.1030 m/z) and diagnostic peak pairs in the extracted ion chromatograms of LC-MS/MS analysis. Using these signatures, we identified citrullination sites in protein arginine deiminase 4 (12 sites) and in fibrinogen (25 sites, two previously unknown). The unique mass spectral features of PTM-dependent proteolytic digest products promise a generalized PTM site-mapping strategy based on a toolbox of such mutant proteases, which are now accessible by laboratory evolution.
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The conformational preferences of γ-lactam and its role in constraining peptide structure
NASA Astrophysics Data System (ADS)
Paul, P. K. C.; Burney, P. A.; Campbell, M. M.; Osguthorpe, D. J.
1990-09-01
The conformational constraints imposed by γ-lactams in peptides have been studied using valence force field energy calculations and flexible geometry maps. It has been found that while cyclisation restrains the Ψ of the lactam, non-bonded interactions contribute to the constraints on ϕ of the lactam. The γ-lactam also affects the (ϕ,Ψ) of the residue after it in a peptide sequence. For an l-lactam, the ring geometry restricts Ψ to about-120°, and ϕ has two minima, the lowest energy around-140° and a higher minimum (5 kcal/mol higher) at 60°, making an l-γ-lactam more favourably accommodated in a near extended conformation than in position 2 of a type II' β-turn. The energy of the ϕ˜+60° minimum can be lowered substantially until it is more favoured than the-140° minimum by progressive substitution of bulkier groups on the amide N of the l-γ-lactam. The (ϕ,Ψ) maps of the residue succeeding a γ-lactam show subtle differences from those of standard N-methylated residues. The dependence of the constraints on the chirality of γ-lactams and N-substituted γ-lactams, in terms of the formation of secondary structures like β-turns is discussed and the comparison of the theoretical conformations with experimental results is highlighted.
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Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed
2014-10-01
One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.
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Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael; Stryhn, Anette; Buus, Søren; Nielsen, Morten
2015-11-01
A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .
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Tang, Chang-Bo; Zhang, Wan-Gang; Wang, Yao-Song; Xing, Lu-Juan; Xu, Xing-Lian; Zhou, Guang-Hong
2016-08-24
Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation.
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Guo, Zuojun; Streu, Kristina; Krilov, Goran; Mohanty, Udayan
2014-06-01
The stabilization of secondary structure is believed to play an important role in the peptide-protein binding interaction. In this study, the α-helical conformation and structural stability of single and double stapled all-hydrocarbon cross-linked p53 peptides when bound and unbound to MDM2 are investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross-linker, the conformation of the double bond in the alkene bridge, and the length of the bridge, to the relative stability of the α-helix structure. The binding affinity calculations by WaterMap provided over one hundred hydration sites in the MDM2 binding pocket where water density is greater than twice that of the bulk, and the relative value of free energy released by displacing these hydration sites. In agreement with the experimental data, potentials of mean force obtained by weighted histogram analysis methods indicated the order of peptides from lowest to highest binding affinity. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53/MDM2 complex. We hope our efforts can help to further the development of a new generation p53/MDM2 inhibitors that can reactivate the function of p53 as tumor suppressor gene. © 2014 John Wiley & Sons A/S.
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MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.
Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wisniewski, Jacek R; Jun, Wang; Mann, Matthias
2007-01-01
Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.
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Jo, Yun Kee; Seo, Jeong Hyun; Choi, Bong-Hyuk; Kim, Bum Jin; Shin, Hwa Hui; Hwang, Byeong Hee; Cha, Hyung Joon
2014-11-26
During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating strategy based on the fusion of MAP to a silver-binding peptide, which can synthesize silver nanoparticles having broad antibacterial activity. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface under mild condition. The biosynthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria and also revealed good cytocompatibility with mammalian cells. In this coating strategy, MAP-silver binding peptide fusion proteins provide hybrid environment incorporating inorganic silver nanoparticle and simultaneously mediate the interaction of silver nanoparticle with surroundings. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.
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Macroporous resin purification of peptides with umami taste from soy sauce.
Zhuang, Mingzhu; Zhao, Mouming; Lin, Lianzhu; Dong, Yi; Chen, Huiping; Feng, Mengying; Sun-Waterhouse, Dongxiao; Su, Guowan
2016-01-01
In this study, the performance and separation characteristics of four macroporous resins for purifying umami peptides from soy sauce were examined. Results showed that the resins could separate the peptides of soy sauce, and the particle diffusion kinetics model was suitable for describing the whole exothermic (ΔH < 43 kJ/mol) adsorption process on the SP-825 and HP-20 resins, while the pseudo-second-order kinetics model accurately described the XAD-16 and HP-2 MGL resins. Furthermore, the adsorption processes of the peptides followed the Freundlich model. The XAD-16 resin was the most effective resin for the enrichment of peptides due to its high adsorption and total desorption capacities. Interestingly, the umami peptides were enriched in the deionized water fraction. This study provides new insights into exploring performance and separation characteristics of macroporous resins on soy sauce, and indicated that peptide may be the contributor to the umami taste in Chinese soy sauce. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Patel, Sunita; Sasidhar, Yellamraju U
2007-10-01
Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.
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Diagnostics and therapy of Alzheimer's disease.
Mikiciuk-Olasik, Elzbieta; Szymański, Paweł; Zurek, Elzbieta
2007-04-01
Alzheimer's Disease (AD) is described as a degenerative disease of the central nervous system characterized by a noticeable cognitive decline defined by a loss of memory and learning ability, together with a reduced ability to perform basic activities of daily living. In the brain of an AD patients is the dramatic decrease in cholinergic innervation in the cortex and hippocampus due to the loss of neurons in the basal forebrain. The above findings led to the development of the cholinergic hypothesis, which proposes that the cognitive loss associated with AD is related to decreased cortical cholinergic neurotransmission. In brain of Alzheimer's patient's one ascertained presence of neuritic plaques containing the beta-amyloid peptide and protein tau. Biochemical and genetics studies implicated a central role for beta-amyloid in the pathological cascade of events in AD. The most therapeutic strategies in AD have been directed to two main targets: the beta-amyloid peptide and the cholinergic neurotransmission. The first approach is to act on the amyloid precursor protein (APP) processing. The second main approach is to slow of decline of neuronal degeneration or increasing cholinergic transmission. Diagnosis of AD is very difficult and to date no specific diagnostic tests of the disease are available. Intellectual function testing to determine the degree of cognitive status during routine medical examination is a useful supplementary method of diagnosing dementia. The permissible result, come down from radiopharmacy, which is an integral part of a nuclear medicine. A radiopharmaceutical may be defined as a pharmaceutical substance containing radioactive atoms. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are capable of mapping the distribution of radionuclides in three dimensions, producing maps of brain biochemical and physiological processes. The techniques are reasonably sensitive and specific in differentiating AD from other dementias.
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Griaud, François; Denefeld, Blandine; Lang, Manuel; Hensinger, Héloïse; Haberl, Peter; Berg, Matthias
2017-01-01
ABSTRACT Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner. PMID:28379786
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Shieh, H M; Bass, R T; Wang, B S; Corbett, M J; Buckwalter, B L
1995-04-01
In this study, the epitope of a murine PS-7.6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70-95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7.6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7.6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70-95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75-90) was the optimal sequence recognized by PS-7.6 mAb. Sequential alanine substitution of each residue of pGH(75-90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7.6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75-95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i + 3 (i + 7) of the critical amino acids appears consistent with PS-7.6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7.6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals.
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Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod; Coin, Irene
2017-01-01
The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors. DOI: http://dx.doi.org/10.7554/eLife.27711.001 PMID:28771403
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McLaughlin, Kerry A; Richardson, Carolyn C; Williams, Stefan; Bonifacio, Ezio; Morgan, Diana; Feltbower, Richard G; Powell, Michael; Rees Smith, Bernard; Furmaniak, Jadwiga; Christie, Michael R
2015-10-01
Diversification of autoimmunity to islet autoantigens is critical for progression to Type 1 diabetes. B-cells participate in diversification by modifying antigen processing, thereby influencing which peptides are presented to T-cells. In Type 1 diabetes, JM antibodies are associated with T-cell responses to PTP domain peptides. We investigated whether this is the consequence of close structural alignment of JM and PTP domain determinants on IA-2. Fab fragments of IA-2 antibodies with epitopes mapped to the JM domain blocked IA-2 binding of antibodies that recognise epitopes in the IA-2 PTP domain. Peptides from both the JM and PTP domains were protected from degradation during proteolysis of JM antibody:IA-2 complexes and included those representing major T-cell determinants in Type 1 diabetes. The results demonstrate close structural relationships between JM and PTP domain epitopes on IA-2. Stabilisation of PTP domain peptides during proteolysis in JM-specific B-cells may explain determinant spreading in IA-2 autoimmunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Szymula, Agnieszka; Rosenthal, Jacob; Szczerba, Barbara M; Bagavant, Harini; Fu, Shu Man; Deshmukh, Umesh S.
2014-01-01
This study was undertaken to test the hypothesis that Sjogren’s syndrome Antigen A (SSA)/Ro60-reactive T cells are activated by peptides originating from oral and gut bacteria. T cell hybridomas generated from HLA-DR3 transgenic mice recognized 3 regions on Ro60, with core epitopes mapped to amino acids 228-238, 246-256 and 371-381. BLAST analysis identified several mimicry peptides, originating from human oral, intestinal, skin and vaginal bacteria, as well as environmental bacteria. Amongst these, a peptide from the von Willebrand factor type A domain protein (vWFA) from the oral microbe Capnocytphaga ochracea was the most potent activator. Further, Ro60-reactive T cells were activated by recombinant vWFA protein and whole E. coli expressing this protein. These results demonstrate that peptides derived from normal human microbiota can activate Ro60-reactive T cells. Thus, immune responses to commensal microbiota and opportunistic pathogens should be explored as potential triggers for initiating autoimmunity in SLE and Sjögren’s syndrome. PMID:24576620
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Pandey, V.N.; Modak, M.J.
Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with (/sup 32/P)ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of (/sup 32/P)ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identicalmore » with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide. The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.« less
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Aletras, A; Barlos, K; Gatos, D; Koutsogianni, S; Mamos, P
1995-05-01
N alpha-9-Fluorenylmethoxycarbonyl-N epsilon-4=methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N epsilon-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.
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Biopharmaceuticals: From peptide to drug
NASA Astrophysics Data System (ADS)
Hannappel, Margarete
2017-08-01
Biologics are therapeutic proteins or peptides that are produced by means of biological processes within living organisms and cells. They are highly specific molecules and play a crucial role as therapeutics for the treatment of severe and chronic diseases (e.g. cancer, rheumatoid arthritis, diabetes, autoimmune disorders). The development of new biologics and biologics-based drugs gains more and more importance in the fight against various diseases. A short overview on biotherapeutical drug development is given. Cone snails are a large group of poisonous, predatory sea snails with more than 700 species. They use a very powerful venom which rapidly inactivates and paralyzes their prey. Most bioactive venom components are small peptides (conotoxins, conopeptides) which are precisely directed towards a specific target (e.g. ion channel, receptors). Due to their small size, their precision and speed of action, naturally occurring cone snail venom peptides represent an attractive source for the identification and design of novel biological drug entities. The Jagna cone snail project is an encouraging initiative to map the ecological variety of cone snails around the island of Bohol (Philippines) and to conserve the biological information for potential future application.
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USDA-ARS?s Scientific Manuscript database
Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide, yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis and...
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Artificial intelligence systems based on texture descriptors for vaccine development.
Nanni, Loris; Brahnam, Sheryl; Lumini, Alessandra
2011-02-01
The aim of this work is to analyze and compare several feature extraction methods for peptide classification that are based on the calculation of texture descriptors starting from a matrix representation of the peptide. This texture-based representation of the peptide is then used to train a support vector machine classifier. In our experiments, the best results are obtained using local binary patterns variants and the discrete cosine transform with selected coefficients. These results are better than those previously reported that employed texture descriptors for peptide representation. In addition, we perform experiments that combine standard approaches based on amino acid sequence. The experimental section reports several tests performed on a vaccine dataset for the prediction of peptides that bind human leukocyte antigens and on a human immunodeficiency virus (HIV-1). Experimental results confirm the usefulness of our novel descriptors. The matlab implementation of our approaches is available at http://bias.csr.unibo.it/nanni/TexturePeptide.zip.
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Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara
2016-01-01
Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692
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Cancer Osaka thyroid (Cot) phosphorylates Polo-like kinase (PLK1) at Ser137 but not at Thr210.
Wu, Binhui; Jiang, Ping; Mu, Yuguang; Wilmouth, Rupert C
2009-12-01
Cancer Osaka thyroid (Cot) is a proto-oncogenic kinase which belongs to the MAP3K family. A peptide-based substrate screening assay revealed that Cot has the ability to phosphorylate Polo-like kinase 1 (Plk1) at Ser137. Kinase assays with intact Plk1 and peptides surrounding Ser137 and Thr210 indicated further that Cot phosphorylates Ser137 but not Thr210. Additional support came from 3D peptide structure prediction and Cot-Plk1 interaction modeling. In vivo experiments demonstrated that wild type Cot, but not a kinase-dead mutant, has the ability to phosphorylate Ser137. Knockdown of Cot in Hela showed a reduction in the level of phosphorylation of Ser137. These results imply for the first time that Cot might be an upstream kinase of Plk1 and suggest a new mechanism for the regulation of the cellular function of Plk1.
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Canterbury, Jesse D.; Merrihew, Gennifer E.; Goodlett, David R.; MacCoss, Michael J.; Shaffer, Scott A.
2015-01-01
A common strategy in mass spectrometry analyses of complex protein mixtures is to digest the proteins to peptides, separate the peptides by microcapillary liquid chromatography and collect tandem mass spectra (MS/MS) on the eluting, complex peptide mixtures, a process commonly termed “shotgun proteomics”. For years, the most common way of data collection was via data-dependent acquisition (DDA), a process driven by an automated instrument control routine that directs MS/MS acquisition from the highest abundant signals to the lowest, a process often leaving lower abundant signals unanalyzed and therefore unidentified in the experiment. Advances in both instrumentation duty cycle and sensitivity allow DDA to probe to lower peptide abundance and therefore enable mapping proteomes to a more significant depth. An alternative to acquiring data by DDA is by data-independent acquisition (DIA), in which a specified range in m/z is fragmented without regard to prioritization of a precursor ion or its relative abundance in the mass spectrum. As a consequence, DIA acquisition potentially offers more comprehensive analysis of peptides than DDA and in principle can yield tandem mass spectra of all ionized molecules following their conversion to the gas-phase. In this work, we evaluate both DDA and DIA on three different linear ion trap instruments: an LTQ, an LTQ modified in-house with an electrodynamic ion funnel, and an LTQ-Velos. These instruments were chosen as they are representative of both older (LTQ) and newer (LTQ-Velos) ion trap designs i.e., linear ion trap and dual ion traps, respectively, and allow direct comparison of peptide identification using both DDA and DIA analysis. Further, as the LTQ-Velos has an improved “S-lens” ion guide in the high-pressure region to improve ion flux, we found it logical to determine if the former LTQ model could be leveraged by improving sensitivity by modifying with an electrodynamic ion guide of significantly different design to the S-lens. We find that the ion funnel enabled LTQ identifies more proteins in the insoluble fraction of a yeast lysate than the other two instruments in DIA mode, while the faster scanning LTQ-Velos performs better in DDA mode. We explore reasons for these results, including differences in scan speed, source ion optics, and linear ion trap design. PMID:25261218
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Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten
2017-01-01
Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.
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Shao, Wenguang; Pedrioli, Patrick G A; Wolski, Witold; Scurtescu, Cristian; Schmid, Emanuel; Vizcaíno, Juan A; Courcelles, Mathieu; Schuster, Heiko; Kowalewski, Daniel; Marino, Fabio; Arlehamn, Cecilia S L; Vaughan, Kerrie; Peters, Bjoern; Sette, Alessandro; Ottenhoff, Tom H M; Meijgaarden, Krista E; Nieuwenhuizen, Natalie; Kaufmann, Stefan H E; Schlapbach, Ralph; Castle, John C; Nesvizhskii, Alexey I; Nielsen, Morten; Deutsch, Eric W; Campbell, David S; Moritz, Robert L; Zubarev, Roman A; Ytterberg, Anders Jimmy; Purcell, Anthony W; Marcilla, Miguel; Paradela, Alberto; Wang, Qi; Costello, Catherine E; Ternette, Nicola; van Veelen, Peter A; van Els, Cécile A C M; Heck, Albert J R; de Souza, Gustavo A; Sollid, Ludvig M; Admon, Arie; Stevanovic, Stefan; Rammensee, Hans-Georg; Thibault, Pierre; Perreault, Claude; Bassani-Sternberg, Michal; Aebersold, Ruedi; Caron, Etienne
2018-01-04
Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren
2017-01-01
Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436
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Mapping the conformational free energy of aspartic acid in the gas phase and in aqueous solution.
Comitani, Federico; Rossi, Kevin; Ceriotti, Michele; Sanz, M Eugenia; Molteni, Carla
2017-04-14
The conformational free energy landscape of aspartic acid, a proteogenic amino acid involved in a wide variety of biological functions, was investigated as an example of the complexity that multiple rotatable bonds produce even in relatively simple molecules. To efficiently explore such a landscape, this molecule was studied in the neutral and zwitterionic forms, in the gas phase and in water solution, by means of molecular dynamics and the enhanced sampling method metadynamics with classical force-fields. Multi-dimensional free energy landscapes were reduced to bi-dimensional maps through the non-linear dimensionality reduction algorithm sketch-map to identify the energetically stable conformers and their interconnection paths. Quantum chemical calculations were then performed on the minimum free energy structures. Our procedure returned the low energy conformations observed experimentally in the gas phase with rotational spectroscopy [M. E. Sanz et al., Phys. Chem. Chem. Phys. 12, 3573 (2010)]. Moreover, it provided information on higher energy conformers not accessible to experiments and on the conformers in water. The comparison between different force-fields and quantum chemical data highlighted the importance of the underlying potential energy surface to accurately capture energy rankings. The combination of force-field based metadynamics, sketch-map analysis, and quantum chemical calculations was able to produce an exhaustive conformational exploration in a range of significant free energies that complements the experimental data. Similar protocols can be applied to larger peptides with complex conformational landscapes and would greatly benefit from the next generation of accurate force-fields.
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Mapping the conformational free energy of aspartic acid in the gas phase and in aqueous solution
NASA Astrophysics Data System (ADS)
Comitani, Federico; Rossi, Kevin; Ceriotti, Michele; Sanz, M. Eugenia; Molteni, Carla
2017-04-01
The conformational free energy landscape of aspartic acid, a proteogenic amino acid involved in a wide variety of biological functions, was investigated as an example of the complexity that multiple rotatable bonds produce even in relatively simple molecules. To efficiently explore such a landscape, this molecule was studied in the neutral and zwitterionic forms, in the gas phase and in water solution, by means of molecular dynamics and the enhanced sampling method metadynamics with classical force-fields. Multi-dimensional free energy landscapes were reduced to bi-dimensional maps through the non-linear dimensionality reduction algorithm sketch-map to identify the energetically stable conformers and their interconnection paths. Quantum chemical calculations were then performed on the minimum free energy structures. Our procedure returned the low energy conformations observed experimentally in the gas phase with rotational spectroscopy [M. E. Sanz et al., Phys. Chem. Chem. Phys. 12, 3573 (2010)]. Moreover, it provided information on higher energy conformers not accessible to experiments and on the conformers in water. The comparison between different force-fields and quantum chemical data highlighted the importance of the underlying potential energy surface to accurately capture energy rankings. The combination of force-field based metadynamics, sketch-map analysis, and quantum chemical calculations was able to produce an exhaustive conformational exploration in a range of significant free energies that complements the experimental data. Similar protocols can be applied to larger peptides with complex conformational landscapes and would greatly benefit from the next generation of accurate force-fields.
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PDB-Explorer: a web-based interactive map of the protein data bank in shape space.
Jin, Xian; Awale, Mahendra; Zasso, Michaël; Kostro, Daniel; Patiny, Luc; Reymond, Jean-Louis
2015-10-23
The RCSB Protein Data Bank (PDB) provides public access to experimentally determined 3D-structures of biological macromolecules (proteins, peptides and nucleic acids). While various tools are available to explore the PDB, options to access the global structural diversity of the entire PDB and to perceive relationships between PDB structures remain very limited. A 136-dimensional atom pair 3D-fingerprint for proteins (3DP) counting categorized atom pairs at increasing through-space distances was designed to represent the molecular shape of PDB-entries. Nearest neighbor searches examples were reported exemplifying the ability of 3DP-similarity to identify closely related biomolecules from small peptides to enzyme and large multiprotein complexes such as virus particles. The principle component analysis was used to obtain the visualization of PDB in 3DP-space. The 3DP property space groups proteins and protein assemblies according to their 3D-shape similarity, yet shows exquisite ability to distinguish between closely related structures. An interactive website called PDB-Explorer is presented featuring a color-coded interactive map of PDB in 3DP-space. Each pixel of the map contains one or more PDB-entries which are directly visualized as ribbon diagrams when the pixel is selected. The PDB-Explorer website allows performing 3DP-nearest neighbor searches of any PDB-entry or of any structure uploaded as protein-type PDB file. All functionalities on the website are implemented in JavaScript in a platform-independent manner and draw data from a server that is updated daily with the latest PDB additions, ensuring complete and up-to-date coverage. The essentially instantaneous 3DP-similarity search with the PDB-Explorer provides results comparable to those of much slower 3D-alignment algorithms, and automatically clusters proteins from the same superfamilies in tight groups. A chemical space classification of PDB based on molecular shape was obtained using a new atom-pair 3D-fingerprint for proteins and implemented in a web-based database exploration tool comprising an interactive color-coded map of the PDB chemical space and a nearest neighbor search tool. The PDB-Explorer website is freely available at www.cheminfo.org/pdbexplorer and represents an unprecedented opportunity to interactively visualize and explore the structural diversity of the PDB. ᅟ
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Cambon, K; Venero, C; Berezin, V; Bock, E; Sandi, C
2003-01-01
The neural cell adhesion molecule (NCAM) plays a key role in synaptic plasticity and memory formation. We have recently developed a synthetic peptide, termed C3d, which, through the binding to the first, N-terminal immunoglobulin-like (Ig) module in the extracellular portion of NCAM, has been shown to promote neurite outgrowth and synapse formation in vitro, and to interfere with passive avoidance memory in rats in vivo. In this study, we investigated whether the i.c.v. administration of C3d, either 5.5 h after or 2 days before training, could be effective to modulate the strength at which emotional memory for aversive situations is established into a long-term memory. The effects of the peptide were evaluated in adult male Wistar rats trained in the contextual fear conditioning task. The results indicated that C3d significantly reduced the subsequent long-term retention of the conditioned fear response when administered 5.5 h post-training, as indicated by retention tests performed 2-3 and 7 days post-training. However, this treatment failed to influence conditioning for this task when injected 2 days pre-training. Additional experiments showed that C3d did not influence the emotional or locomotor behaviour of the animals, when tested in the open field task. Furthermore, hippocampal levels of microtubule-associated protein 2 (MAP2), Synaptophysin and NCAM were found unchanged when evaluated by enzyme-linked immunosorbent assay in crude synaptosomal preparations 2 days after peptide i.c.v. injection. Therefore, post-training injection of this synthetic peptide was efficient to attenuate the strength at which memory for contextual fear conditioning was enduringly stored, whilst it did not affect the acquisition of new memories. In addition to further support the view that NCAM is critically involved in memory consolidation, the current findings suggest that the NCAM IgI module is a potential target for the development of therapeutic drugs capable to reduce the cognitive impact induced by exposure to intensive stress experiences.
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Santona, Antonella; Carta, Franco; Fraghí, Peppinetta; Turrini, Franco
2002-01-01
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection. PMID:11748179
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Disertori, Marcello; Quintarelli, Silvia; Grasso, Maurizia; Pilotto, Andrea; Narula, Nupoor; Favalli, Valentina; Canclini, Camilla; Diegoli, Marta; Mazzola, Silvia; Marini, Massimiliano; Del Greco, Maurizio; Bonmassari, Roberto; Masè, Michela; Ravelli, Flavia; Specchia, Claudia; Arbustini, Eloisa
2013-02-01
Atrial dilatation and atrial standstill are etiologically heterogeneous phenotypes with poorly defined nosology. In 1983, we described 8-years follow-up of atrial dilatation with standstill evolution in 8 patients from 3 families. We later identified 5 additional patients with identical phenotypes: 1 member of the largest original family and 4 unrelated to the 3 original families. All families are from the same geographic area in Northeast Italy. We followed up the 13 patients for up to 37 years, extended the clinical investigation and monitoring to living relatives, and investigated the genetic basis of the disease. The disease was characterized by: (1) clinical onset in adulthood; (2) biatrial dilatation up to giant size; (3) early supraventricular arrhythmias with progressive loss of atrial electric activity to atrial standstill; (4) thromboembolic complications; and (5) stable, normal left ventricular function and New York Heart Association functional class during the long-term course of the disease. By linkage analysis, we mapped a locus at 1p36.22 containing the Natriuretic Peptide Precursor A gene. By sequencing Natriuretic Peptide Precursor A, we identified a homozygous missense mutation (p.Arg150Gln) in all living affected individuals of the 6 families. All patients showed low serum levels of atrial natriuretic peptide. Heterozygous mutation carriers were healthy and demonstrated normal levels of atrial natriuretic peptide. Autosomal recessive atrial dilated cardiomyopathy is a rare disease associated with homozygous mutation of the Natriuretic Peptide Precursor A gene and characterized by extreme atrial dilatation with standstill evolution, thromboembolic risk, preserved left ventricular function, and severely decreased levels of atrial natriuretic peptide.
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Infrared Multiphoton Dissociation for Quantitative Shotgun Proteomics
Ledvina, Aaron R.; Lee, M. Violet; McAlister, Graeme C.; Westphall, Michael S.; Coon, Joshua J.
2012-01-01
We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low pressure trap of a dual-cell quadrupole linear ion trap (dual cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly outperforms resonant excitation CAD for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT RF amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass-to-charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides. PMID:22480380
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MzJava: An open source library for mass spectrometry data processing.
Horlacher, Oliver; Nikitin, Frederic; Alocci, Davide; Mariethoz, Julien; Müller, Markus; Lisacek, Frederique
2015-11-03
Mass spectrometry (MS) is a widely used and evolving technique for the high-throughput identification of molecules in biological samples. The need for sharing and reuse of code among bioinformaticians working with MS data prompted the design and implementation of MzJava, an open-source Java Application Programming Interface (API) for MS related data processing. MzJava provides data structures and algorithms for representing and processing mass spectra and their associated biological molecules, such as metabolites, glycans and peptides. MzJava includes functionality to perform mass calculation, peak processing (e.g. centroiding, filtering, transforming), spectrum alignment and clustering, protein digestion, fragmentation of peptides and glycans as well as scoring functions for spectrum-spectrum and peptide/glycan-spectrum matches. For data import and export MzJava implements readers and writers for commonly used data formats. For many classes support for the Hadoop MapReduce (hadoop.apache.org) and Apache Spark (spark.apache.org) frameworks for cluster computing was implemented. The library has been developed applying best practices of software engineering. To ensure that MzJava contains code that is correct and easy to use the library's API was carefully designed and thoroughly tested. MzJava is an open-source project distributed under the AGPL v3.0 licence. MzJava requires Java 1.7 or higher. Binaries, source code and documentation can be downloaded from http://mzjava.expasy.org and https://bitbucket.org/sib-pig/mzjava. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.
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Accelerating String Set Matching in FPGA Hardware for Bioinformatics Research
Dandass, Yoginder S; Burgess, Shane C; Lawrence, Mark; Bridges, Susan M
2008-01-01
Background This paper describes techniques for accelerating the performance of the string set matching problem with particular emphasis on applications in computational proteomics. The process of matching peptide sequences against a genome translated in six reading frames is part of a proteogenomic mapping pipeline that is used as a case-study. The Aho-Corasick algorithm is adapted for execution in field programmable gate array (FPGA) devices in a manner that optimizes space and performance. In this approach, the traditional Aho-Corasick finite state machine (FSM) is split into smaller FSMs, operating in parallel, each of which matches up to 20 peptides in the input translated genome. Each of the smaller FSMs is further divided into five simpler FSMs such that each simple FSM operates on a single bit position in the input (five bits are sufficient for representing all amino acids and special symbols in protein sequences). Results This bit-split organization of the Aho-Corasick implementation enables efficient utilization of the limited random access memory (RAM) resources available in typical FPGAs. The use of on-chip RAM as opposed to FPGA logic resources for FSM implementation also enables rapid reconfiguration of the FPGA without the place and routing delays associated with complex digital designs. Conclusion Experimental results show storage efficiencies of over 80% for several data sets. Furthermore, the FPGA implementation executing at 100 MHz is nearly 20 times faster than an implementation of the traditional Aho-Corasick algorithm executing on a 2.67 GHz workstation. PMID:18412963
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Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T
2017-03-17
Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.
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Exploration of the Medicinal Peptide Space.
Gevaert, Bert; Stalmans, Sofie; Wynendaele, Evelien; Taevernier, Lien; Bracke, Nathalie; D'Hondt, Matthias; De Spiegeleer, Bart
2016-01-01
The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs.
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Yu, Yue; Liu, Hongwei; Tu, Maolin; Qiao, Meiling; Wang, Zhenyu; Du, Ming
2017-12-01
Ruditapes philippinarum is nutrient-rich and widely-distributed, but little attention has been paid to the identification and characterization of the bioactive peptides in the bivalve. In the present study, we evaluated the peptides of the R. philippinarum that were enzymolysised by trypsin using a combination of ultra-performance liquid chromatography separation and electrospray ionization quadrupole time-of-flight tandem mass spectrometry, followed by data processing and sequence-similarity database searching. The potential allergenicity of the peptides was assessed in silico. The enzymolysis was performed under the conditions: E:S 3:100 (w/w), pH 9.0, 45 °C for 4 h. After separation and detection, the Swiss-Prot database and a Ruditapes philippinarum sequence database were used: 966 unique peptides were identified by non-error tolerant database searching; 173 peptides matching 55 precursor proteins comprised highly conserved cytoskeleton proteins. The remaining 793 peptides were identified from the R. philippinarum sequence database. The results showed that 510 peptides were labeled as allergens and 31 peptides were potential allergens; 425 peptides were predicted to be nonallergenic. The abundant peptide information contributes to further investigations of the structure and potential function of R. philippinarum. Additional in vitro studies are required to demonstrate and ensure the correct production of the hydrolysates for use in the food industry with respect to R. philippinarum. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
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Plaviak, Alexandra; Osburn, Sandra; Patterson, Khiry; van Stipdonk, Michael J
2016-01-15
Experiments were performed to probe the creation of apparent even-electron, [M-H](+) ions by CID of Ag-cationized peptides with N-terminal imine groups (Schiff bases). Imine-modified peptides were prepared using condensation reactions with aldehydes. Ag(+) -cationized precursors were generated by electrospray ionization (ESI). Tandem mass spectrometry (MS(n) ) and collision-induced dissociation (CID) were performed using a linear ion trap mass spectrometer. Loss of AgH from peptide [M + Ag](+) ions, at the MS/MS stage, creates closed-shell [M-H](+) ions from imine-modified peptides. Isotope labeling unambiguously identifies the imine C-H group as the source of H eliminated in AgH. Subsequent CID of the [M-H](+) ions generated sequence ions that are analogous to those produced from [M + H](+) ions of the imine-modified peptides. Experiments show (a) formation of novel even-electron peptide cations by CID and (b) the extent to which sequence ions (conventional b, a and y ions) are generated from peptides with fixed charge site and thus lacking a conventional mobile proton. Copyright © 2015 John Wiley & Sons, Ltd.
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Peptide de novo sequencing of mixture tandem mass spectra
Hotta, Stéphanie Yuki Kolbeck; Verano‐Braga, Thiago; Kjeldsen, Frank
2016-01-01
The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co‐isolation and thus prone to false identifications. The deconvolution approach matched complementary b‐, y‐ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co‐isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. PMID:27329701
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Zolg, Daniel Paul; Wilhelm, Mathias; Yu, Peng; Knaute, Tobias; Zerweck, Johannes; Wenschuh, Holger; Reimer, Ulf; Schnatbaum, Karsten; Kuster, Bernhard
2017-11-01
Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike-in peptides are not yet systematically used as internal standards in bottom-up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC-MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Ludwig, Katelyn R.; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.; Hummon, Amanda B.
2015-01-01
Ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome analysis. Alternatively, capillary zone electrophoresis (CZE) - ESI-MS/MS has great potential for phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides. In this work, we systematically compared UPLC-MS/MS and CZE-MS/MS for phosphorylated peptide identifications (IDs) using an enriched phosphoproteome from the MCF-10A cell line. When the sample loading amount of UPLC was 10 times higher than that of CZE (2 μg vs. 200 ng), UPLC generated more phosphorylated peptide IDs than CZE (3,313 vs. 1,783). However, when the same sample loading amounts were used for CZE and UPLC (2–200 ng), CZE-MS/MS consistently and significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. This superior performance is most likely due to the higher peptide intensity generated by CZE-MS/MS. More importantly, compared with UPLC data from 2 μg sample, CZE-MS/MS can identify over 500 unique phosphorylated peptides from 200 ng sample, suggesting that CZE and UPLC are complementary for phosphorylated peptide IDs. With further improved loading capacity via a dynamic pH junction method, 2,313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. This number of phosphorylated peptide IDs is over one order of magnitude higher than the number of phosphorylated peptide IDs previously reported by single-shot CZE-MS/MS. PMID:26399161
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Identification of tissue-specific targeting peptide
NASA Astrophysics Data System (ADS)
Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun
2012-11-01
Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.
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Mindaye, S T; Spiric, J; David, N A; Rabin, R L; Slater, J E
2017-12-01
German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range (r > 0.99, 0.01-1384 fmol/μL), and sensitive (LLOD and LLOQ <1 fmol/μL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies. © 2017 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.
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Halimi, Hubert; De Caro, Josiane; Carrière, Frédéric; De Caro, Alain
2005-12-01
Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.
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Ma, Xianyue; Cline, Kenneth
2013-03-01
Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.
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Heat-enhanced peptide synthesis on Teflon-patterned paper.
Deiss, Frédérique; Yang, Yang; Matochko, Wadim L; Derda, Ratmir
2016-06-14
In this report, we describe the methodology for 96 parallel organic syntheses of peptides on Teflon-patterned paper assisted by heating with an infra-red lamp. SPOT synthesis is an important technology for production of peptide arrays on a paper-based support for rapid identification of peptide ligands, epitope mapping, and identification of bio-conjugation reactions. The major drawback of the SPOT synthesis methodology published to-date is suboptimal reaction conversion due to mass transport limitations in the unmixed reaction spot. The technology developed in this report overcomes these problems by changing the environment of the reaction from static to dynamic (flow-through), and further accelerating the reaction by selective heating of the reaction support in contact with activated amino acids. Patterning paper with Teflon allows for droplets of organic solvents to be confined in a zone on the paper array and flow through the paper at a well-defined rate and provide a convenient, power-free setup for flow-through solid-phase synthesis and efficient assembly of peptide arrays. We employed an infra-red (IR) lamp to locally heat the cellulosic support during the flow-through delivery of the reagents to each zone of the paper-based array. We demonstrate that IR-heating in solid phase peptide synthesis shortened the reaction time necessary for amide bond formation down to 3 minutes; in some couplings of alpha amino acids, conversion rates increased up to fifteen folds. The IR-heating improved the assembly of difficult sequences, such as homo-oligomers of all 20 natural amino acids.
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Cacchioli, Antonio; Ravanetti, Francesca; Bagno, Andrea; Dettin, Monica; Gabbi, Carlo
2009-10-01
Peptide and protein exploitation for the biochemical functionalization of biomaterial surfaces allowed fabricating biomimetic devices able to evoke and promote specific and advantageous cell functions in vitro and in vivo. In particular, cell adhesion improvement to support the osseointegration of implantable devices has been thoroughly investigated. This study was aimed at checking the biological activity of the (351-359) human vitronectin precursor (HVP) sequence, mapped on the human vitronectin protein; the peptide was covalently linked to the surface of titanium cylinders, surgically inserted in the femurs of New Zealand white rabbits and analyzed at short experimental time points (4, 9, and 16 days after surgery). To assess the osteogenic activity of the peptide, three vital fluorochromic bone markers were used (calcein green, xylenol orange, and calcein blue) to stain the areas of newly grown bone. Static and dynamic histomorphometric parameters were measured at the bone-implant interface and at different distances from the surface. The biological role of the (351-359)HVP sequence was checked by comparing peptide-grafted samples and controls, analyzing how and how much its effects change with time across the bone regions surrounding the implant surface. The results obtained reveal a major activity of the investigated peptide 4 days after surgery, within the bone region closest to the implant surface, and larger bone to implant contact 9 and 16 days after surgery. Thus, improved primary fixation of endosseous devices can be foreseen, resulting in an increased osteointegration.
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The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization.
Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Bukhari, Tallat H; Stein, Stephen E
2018-04-01
We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.
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Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I
2014-01-01
Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426
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The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization
Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P.; Mirokhin, Yuri A.; Tchekhovskoi, Dmitrii V.; Bukhari, Tallat H.; Stein, Stephen E.
2018-01-01
ABSTRACT We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins. PMID:29425077
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Engineering antimicrobial peptides with improved antimicrobial and hemolytic activities.
Zhao, Jun; Zhao, Chao; Liang, Guizhao; Zhang, Mingzhen; Zheng, Jie
2013-12-23
The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.
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Blocking the association of HDAC4 with MAP1S accelerates autophagy clearance of mutant Huntingtin
Yue, Fei; Li, Wenjiao; Zou, Jing; Chen, Qi; Xu, Guibin; Huang, Hai; Xu, Zhen; Zhang, Sheng; Gallinari, Paola; Wang, Fen; McKeehan, Wallace L.; Liu, Leyuan
2015-01-01
Autophagy controls and executes the turnover of abnormally aggregated proteins. MAP1S interacts with the autophagy marker LC3 and positively regulates autophagy flux. HDAC4 associates with the aggregation-prone mutant huntingtin protein (mHTT) that causes Huntington's disease, and colocalizes with it in cytosolic inclusions. It was suggested HDAC4 interacts with MAP1S in a yeast two-hybrid screening. Here, we found that MAP1S interacts with HDAC4 via a HDAC4-binding domain (HBD). HDAC4 destabilizes MAP1S, suppresses autophagy flux and promotes the accumulation of mHTT aggregates. This occurs by an increase in the deacetylation of the acetylated MAP1S. Either suppression of HDAC4 with siRNA or overexpression of the MAP1S HBD leads to stabilization of MAP1S, activation of autophagy flux and clearance of mHTT aggregates. Therefore, specific interruption of the HDAC4-MAP1S interaction with short peptides or small molecules to enhance autophagy flux may relieve the toxicity of mHTT associated with Huntington's disease and improve symptoms of HD patients. PMID:26540094
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Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests.
Vanhoenacker, Gerd; Vandenheede, Isabel; David, Frank; Sandra, Pat; Sandra, Koen
2015-01-01
Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cation-exchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and (2)D retention time opening interesting perspectives for use in QA/QC testing.
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Reisinger, Florian; Krishna, Ritesh; Ghali, Fawaz; Ríos, Daniel; Hermjakob, Henning; Vizcaíno, Juan Antonio; Jones, Andrew R
2012-03-01
We present a Java application programming interface (API), jmzIdentML, for the Human Proteome Organisation (HUPO) Proteomics Standards Initiative (PSI) mzIdentML standard for peptide and protein identification data. The API combines the power of Java Architecture of XML Binding (JAXB) and an XPath-based random-access indexer to allow a fast and efficient mapping of extensible markup language (XML) elements to Java objects. The internal references in the mzIdentML files are resolved in an on-demand manner, where the whole file is accessed as a random-access swap file, and only the relevant piece of XMLis selected for mapping to its corresponding Java object. The APIis highly efficient in its memory usage and can handle files of arbitrary sizes. The APIfollows the official release of the mzIdentML (version 1.1) specifications and is available in the public domain under a permissive licence at http://www.code.google.com/p/jmzidentml/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Mora Osorio, Camilo Andrés; González Barrios, Andrés Fernando
2016-12-07
Calculation of the Gibbs free energy changes of biological molecules at the oil-water interface is commonly performed with Molecular Dynamics simulations (MD). It is a process that could be performed repeatedly in order to find some molecules of high stability in this medium. Here, an alternative method of calculation has been proposed: a group contribution method (GCM) for peptides based on MD of the twenty classic amino acids to obtain free energy change during the insertion of any peptide chain in water-dodecane interfaces. Multiple MD of the twenty classic amino acids located at the interface of rectangular simulation boxes with a dodecane-water medium were performed. A GCM to calculate the free energy of entire peptides is then proposed. The method uses the summation of the Gibbs free energy of each amino acid adjusted in function of its presence or absence in the chain as well as its hydrophobic characteristics. Validation of the equation was performed with twenty-one peptides all simulated using MD in dodecane-water rectangular boxes in previous work, obtaining an average relative error of 16%.
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Xu, Wan-Xiang; Wang, Jian; Tang, Hai-Ping; He, Ya-Ping; Zhu, Qian-Xi; Gupta, Satish K.; Gu, Shao-Hua; Huang, Qiang; Ji, Chao-Neng; Liu, Ling-Feng; Li, Gui-Ling; Xu, Cong-Jian; Xie, Yi
2016-01-01
To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and ‘universal’ preventive HPV peptide vaccine based on L1 conserved BCEs. PMID:27708433
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Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair
2011-06-01
The continuous regions on botulinum neurotoxin A (BoNT/A) light (L) chain recognized by anti-toxin antibodies (Abs) from mouse, horse and chicken have been mapped. We synthesized a panel of thirty-two 19-residue peptides that overlapped consecutively by 5 residues and encompassed the entire L chain (residues 1-453). Mouse Abs recognized 5 major antigenic regions on the L chain, horse Abs recognized 9 while chicken Abs recognized 8 major antigenic regions. Overall, however, the three host species recognized, to some extent, similar, but not identical, peptides and the levels of Abs directed against a given region varied with the immunized host. Differences in the MHC of the host caused variation in levels of Ab recognition and some epitopes showed right or left frame-shifts among the species. Selected region(s) were also uniquely recognized by one species (e.g., peptide L1 by horse Abs). Mapping of the L chain antigenic regions and the previous localization of the regions on the H chain with the same antisera, has permitted description of the complete antigenic structure of BoNT/A. The locations in the 3-dimensional structure of the antigenic regions of the entire toxin are shown for mouse Abs. In the 3-D structure, the antigenic regions are on the surface of the toxin and when antibodies are bound the enzymatic activity of the light chain is obstructed. Copyright © 2010 Elsevier GmbH. All rights reserved.
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Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia
Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality. © PDA, Inc. 2016.
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Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.
Burns, C; Bristow, A F; Buchheit, K H; Daas, A; Wierer, M; Costanzo, A
2015-01-01
The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.
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Frickenhaus, Stephan; Kannan, Srinivasaraghavan; Zacharias, Martin
2009-02-01
A direct conformational clustering and mapping approach for peptide conformations based on backbone dihedral angles has been developed and applied to compare conformational sampling of Met-enkephalin using two molecular dynamics (MD) methods. Efficient clustering in dihedrals has been achieved by evaluating all combinations resulting from independent clustering of each dihedral angle distribution, thus resolving all conformational substates. In contrast, Cartesian clustering was unable to accurately distinguish between all substates. Projection of clusters on dihedral principal component (PCA) subspaces did not result in efficient separation of highly populated clusters. However, representation in a nonlinear metric by Sammon mapping was able to separate well the 48 highest populated clusters in just two dimensions. In addition, this approach also allowed us to visualize the transition frequencies between clusters efficiently. Significantly, higher transition frequencies between more distinct conformational substates were found for a recently developed biasing-potential replica exchange MD simulation method allowing faster sampling of possible substates compared to conventional MD simulations. Although the number of theoretically possible clusters grows exponentially with peptide length, in practice, the number of clusters is only limited by the sampling size (typically much smaller), and therefore the method is well suited also for large systems. The approach could be useful to rapidly and accurately evaluate conformational sampling during MD simulations, to compare different sampling strategies and eventually to detect kinetic bottlenecks in folding pathways.
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Pacios, Luis F; Tordesillas, Leticia; Cuesta-Herranz, Javier; Compes, Esther; Sánchez-Monge, Rosa; Palacín, Arantxa; Salcedo, Gabriel; Díaz-Perales, Araceli
2008-04-01
Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.
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Xiong, Kan; Zwier, Matthew C.; Myshakina, Nataliya S.; Burger, Virginia M.; Asher, Sanford A.; Chong, Lillian T.
2011-01-01
We report the first experimental measurements of Ramachandran Ψ-angle distributions for intrinsically disordered peptides: the N-terminal peptide fragment of tumor suppressor p53 and its P27 mutant form. To provide atomically detailed views of the conformational distributions, we performed classical, explicit-solvent molecular dynamics simulations on the microsecond timescale. Upon binding its partner protein, MDM2, wild-type p53 peptide adopts an α-helical conformation. Mutation of Pro27 to serine results in the highest affinity yet observed for MDM2-binding of the p53 peptide. Both UV resonance Raman spectroscopy (UVRR) and simulations reveal that the P27S mutation decreases the extent of PPII helical content and increases the probability for conformations that are similar to the α-helical MDM2-bound conformation. In addition, UVRR measurements were performed on peptides that were isotopically labeled at the Leu26 residue preceding the Pro27 in order to determine the conformational distributions of Leu26 in the wild-type and mutant peptides. The UVRR and simulation results are in quantitative agreement in terms of the change in the population of non-PPII conformations involving Leu26 upon mutation of Pro27 to serine. Finally, our simulations reveal that the MDM2-bound conformation of the peptide is significantly populated in both the wild-type and mutant isolated peptide ensembles in their unbound states, suggesting that MDM2 binding of the p53 peptides may involve conformational selection. PMID:21528875
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Quantitative modeling of peptide binding to TAP using support vector machine.
Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A
2010-01-01
The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.
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Hb taradale [beta82(EF6)Lys-->Arg]: a novel mutation at a 2,3-diphosphoglycerate binding site.
Brennan, Stephen O; Sheen, Campbell; Chan, Tim; George, Peter M
2005-01-01
Hb Taradale [beta82(EF6)Lys-->Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys-->Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.
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Kuhlmann, F E; Apffel, A; Fischer, S M; Goldberg, G; Goodley, P C
1995-12-01
Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process "masks" the protonated sample cations from the ESI-MS electric fields by rendering them "neutral. " Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.
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Proteome-wide inference of human endophilin 1-binding peptides.
Wu, Gang; Zhang, Zeng-Li; Fu, Chun-Jiang; Lv, Feng-Lin; Tian, Fei-Fei
2012-10-01
Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of prolinerich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3-peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/ sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3-peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3-peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.
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Enhancing the efficiency of sortase-mediated ligations through nickel-peptide complex formation.
David Row, R; Roark, Travis J; Philip, Marina C; Perkins, Lorena L; Antos, John M
2015-08-14
A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this approach was demonstrated with both peptide and protein substrates.
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Peptide de novo sequencing of mixture tandem mass spectra.
Gorshkov, Vladimir; Hotta, Stéphanie Yuki Kolbeck; Verano-Braga, Thiago; Kjeldsen, Frank
2016-09-01
The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20-35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Aoshi, Taiki; Suzuki, Mina; Uchijima, Masato; Nagata, Toshi; Koide, Yukio
2005-03-01
Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.
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Xu, Bo; Vasile, Silvana; Østergaard, Søren; Paulsson, Johan F; Pruner, Jasna; Åqvist, Johan; Wulff, Birgitte S; Gutiérrez-de-Terán, Hugo; Larhammar, Dan
2018-04-01
Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y 2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment ( 32 TRQRY 36 -amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y 2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln288 6.55 and Tyr219 5.39 , while Gln130 3.32 contributes to interactions with Q 34 in the peptide and T 32 is close to the tip of TM7 in the receptor. This leaves the core, α -helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y 2 system and can be used as a basis for optimization of Y 2 receptor agonists. Copyright © 2018 by The Author(s).
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Hayashi, Kazuhiro; Mochizuki, Mayumi; Nomizu, Motoyoshi; Uchinuma, Eijyu; Yamashina, Shohei; Kadoya, Yuichi
2002-04-01
We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.
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Hecker, Michael; Fitzner, Brit; Wendt, Matthias; Lorenz, Peter; Flechtner, Kristin; Steinbeck, Felix; Schröder, Ina; Thiesen, Hans-Jürgen; Zettl, Uwe Klaus
2016-01-01
Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g. MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (p values <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392–411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS. PMID:26831522
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Hao, Piliang; Ren, Yan; Dutta, Bamaprasad; Sze, Siu Kwan
2013-04-26
ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compared the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionates peptides according to their pI and GRAVY values. These properties remains but becomes less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY values of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP is good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives. For in-depth proteomic analysis of a cell, tissue and plasma, multidimensional liquid chromatography (MDLC) is still necessary to reduce sample complexity for improving analytical dynamic range and proteome coverage. This work conducts a direct comparison of three promising first-dimensional proteome fractionation methods. They are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC. ERLIC is good at analyzing basic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI<4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods. Copyright © 2013 Elsevier B.V. All rights reserved.
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Peptide-based antibody alternatives for biological sensing in austere environments
NASA Astrophysics Data System (ADS)
Coppock, Matthew B.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.
2017-02-01
The most critical component of a biosensor, the biorecognition element, must exhibit high selectivity and strong affinity for a target of interest in operational sensing. Monoclonal antibodies are the current standard reagents for such devices, but their adaptability, manufacturability, and stability greatly limit their effectiveness in fieldable sensors. Peptides have emerged as potential antibody replacements in such applications due to their similar binding performance, extreme chemical and thermal stabilities, and on-demand scalability. In conjunction with modeling capabilities, work at the Army Research Lab focuses on protein catalyzed capture (PCC) agent technology and bacterial display for the discovery of these novel peptide binding reagents. The synthetic, bottom-up PCC agent technology uses an iterative, in situ "click chemistry" approach to produce high performing peptides against specific epitopes translatable to the protein target. Bacterial display allows rapid reagent discovery due to the combination of fast bacterial growth and effective peptide sequence enrichment through multiple rounds of biopanning. Recent advances in both methods are highlighted in regards to the discovery of reagents against Army high priority protein targets for soldier safety, performance, and diagnostics.
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NASA Astrophysics Data System (ADS)
Reppert, Michael; Tokmakoff, Andrei
The structural characterization of intrinsically disordered peptides (IDPs) presents a challenging biophysical problem. Extreme heterogeneity and rapid conformational interconversion make traditional methods difficult to interpret. Due to its ultrafast (ps) shutter speed, Amide I vibrational spectroscopy has received considerable interest as a novel technique to probe IDP structure and dynamics. Historically, Amide I spectroscopy has been limited to delivering global secondary structural information. More recently, however, the method has been adapted to study structure at the local level through incorporation of isotope labels into the protein backbone at specific amide bonds. Thanks to the acute sensitivity of Amide I frequencies to local electrostatic interactions-particularly hydrogen bonds-spectroscopic data on isotope labeled residues directly reports on local peptide conformation. Quantitative information can be extracted using electrostatic frequency maps which translate molecular dynamics trajectories into Amide I spectra for comparison with experiment. Here we present our recent efforts in the development of a rigorous approach to incorporating Amide I spectroscopic restraints into refined molecular dynamics structural ensembles using maximum entropy and related approaches. By combining force field predictions with experimental spectroscopic data, we construct refined structural ensembles for a family of short, strongly disordered, elastin-like peptides in aqueous solution.
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Application of acute maximal exercise to protect orthostatic tolerance after simulated microgravity
NASA Technical Reports Server (NTRS)
Engelke, K. A.; Doerr, D. F.; Crandall, C. G.; Convertino, V. A.
1996-01-01
We tested the hypothesis that one bout of maximal exercise performed at the conclusion of prolonged simulated microgravity would improve blood pressure stability during an orthostatic challenge. Heart rate (HR), mean arterial blood pressure (MAP), norepinephrine (NE), epinephrine (E), arginine vasopressin (AVP), plasma renin activity (PRA), atrial natriuretic peptide (ANP), cardiac output (Q), forearm vascular resistance (FVR), and changes in leg volume were measured during lower body negative pressure (LBNP) to presyncope in seven subjects immediately prior to reambulation from 16 days of 6 degrees head-down tilt (HDT) under two experimental conditions: 1) after maximal supine cycle ergometry performed 24 h before returning to the upright posture (exercise) and 2) without exercise (control). After HDT, the reduction of LBNP tolerance time from pre-HDT levels was greater (P = 0.041) in the control condition (-2.0 +/- 0.2 min) compared with the exercise condition (-0.4 +/- 0.2 min). At presyncope after HDT, FVR and NE were higher (P < 0.05) after exercise compared with control, whereas MAP, HR, E, AVP, PRA, ANP, and leg volume were similar in both conditions. Plasma volume (PV) and carotid-cardiac baroreflex sensitivity were reduced after control HDT, but were restored by the exercise treatment. Maintenance of orthostatic tolerance by application of acute intense exercise after 16 days of simulated microgravity was associated with greater circulating levels of NE, vasoconstriction, Q, baroreflex sensitivity, and PV.
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Wang, Zhi-Xiang; Duan, Yong
2004-11-15
The effects of solvation on the conformations and energies of alanine dipeptide (AD) have been studied by ab initio calculations up to MP2/cc-pVTZ//MP2/6-31G**, utilizing the polarizable continuum model (PCM) to mimic solvation effects. The energy surfaces in the gas phase, ether, and water bear similar topological features carved by the steric hindrance, but the details differ significantly due to the solvent effects. The gas-phase energy map is qualitatively consistent with the Ramachandran plot showing seven energy minima. With respect to the gas-phase map, the significant changes of the aqueous map include (1) the expanded low-energy regions, (2) the emergence of an energy barrier between C5-beta and alpha(R)-beta(2) regions, (3) a clearly pronounced alpha(R) minimum, a new beta-conformer, and the disappearance of the gas-phase global minimum, and (4) the shift of the dominant region in LEII from the gas-phase C7(ax) region to the alpha(L) region. These changes bring the map in water to be much closer to the Ramachandran plot than the gas-phase map. The solvent effects on the geometries include the elongation of the exposed N-H and C=O bonds, the shortening of the buried HN--CO peptide bonds, and the enhanced planarity of the peptide bonds. The energy surface in ether has features similar to those both in the gas phase and in water. The free energy order computed in the gas phase and in ether is in good agreement with experimental studies that concluded that C5 and C7(eq) are the dominant species in both the gas phase and nonpolar solvents. The free energy order in water is consistent with the experimental observation that the dominant C7(eq) in the nonpolar solvent was largely replaced by P(II)-like (i.e., beta) and alpha(R) in the strong polar solvents. Based on calculations on AD + 4H(2)O and other AD-water clusters, we suggest that explicit water-AD interactions may distort C5 and beta (or alpha(R) and beta) to an intermediate conformation. Our analysis also shows that the PCM calculations at the MP2/cc-pVTZ//MP2/6-31G** level give good descriptions to the bulk solvent polarization effect. The results presented in this article should be of sufficient quality to characterize the peptide bonds in the gas phase and solvents. The energy surfaces may serve as the basis for developing of strategies enabling the inclusion of solvent polarization in the force field.
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Hydra: a scalable proteomic search engine which utilizes the Hadoop distributed computing framework
2012-01-01
Background For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. Results We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. Conclusion The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources. PMID:23216909
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Hydra: a scalable proteomic search engine which utilizes the Hadoop distributed computing framework.
Lewis, Steven; Csordas, Attila; Killcoyne, Sarah; Hermjakob, Henning; Hoopmann, Michael R; Moritz, Robert L; Deutsch, Eric W; Boyle, John
2012-12-05
For shotgun mass spectrometry based proteomics the most computationally expensive step is in matching the spectra against an increasingly large database of sequences and their post-translational modifications with known masses. Each mass spectrometer can generate data at an astonishingly high rate, and the scope of what is searched for is continually increasing. Therefore solutions for improving our ability to perform these searches are needed. We present a sequence database search engine that is specifically designed to run efficiently on the Hadoop MapReduce distributed computing framework. The search engine implements the K-score algorithm, generating comparable output for the same input files as the original implementation. The scalability of the system is shown, and the architecture required for the development of such distributed processing is discussed. The software is scalable in its ability to handle a large peptide database, numerous modifications and large numbers of spectra. Performance scales with the number of processors in the cluster, allowing throughput to expand with the available resources.
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9-Fluorenylmethyloxycarbonyl/ tbutyl-based convergent protein synthesis.
Barlos, K; Gatos, D
1999-01-01
Besides linear solid phase peptide synthesis, segment condensation in solution and chemical ligation, convergent peptide synthesis (CPS) was developed in order to enable the efficient preparation of complex peptides and small proteins. According to this synthetic strategy, solid phase synthesized and suitably protected peptide fragments corresponding to the entire peptide/protein-sequence are condensed on a solid support or in solution, to the target protein. This review summarizes CPS performed utilizing the mild 9-fluorenylmethyloxycarbonyl/tbutyloxycarbonyl-based protecting scheme for the amino acids. Copyright 1999 John Wiley & Sons, Inc.
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Why are they missing? : Bioinformatics characterization of missing human proteins.
Elguoshy, Amr; Magdeldin, Sameh; Xu, Bo; Hirao, Yoshitoshi; Zhang, Ying; Kinoshita, Naohiko; Takisawa, Yusuke; Nameta, Masaaki; Yamamoto, Keiko; El-Refy, Ali; El-Fiky, Fawzy; Yamamoto, Tadashi
2016-10-21
NeXtProt is a web-based protein knowledge platform that supports research on human proteins. NeXtProt (release 2015-04-28) lists 20,060 proteins, among them, 3373 canonical proteins (16.8%) lack credible experimental evidence at protein level (PE2:PE5). Therefore, they are considered as "missing proteins". A comprehensive bioinformatic workflow has been proposed to analyze these "missing" proteins. The aims of current study were to analyze physicochemical properties, existence and distribution of the tryptic cleavage sites, and to pinpoint the signature peptides of the missing proteins. Our findings showed that 23.7% of missing proteins were hydrophobic proteins possessing transmembrane domains (TMD). Also, forty missing entries generate tryptic peptides were either out of mass detection range (>30aa) or mapped to different proteins (<9aa). Additionally, 21% of missing entries didn't generate any unique tryptic peptides. In silico endopeptidase combination strategy increased the possibility of missing proteins identification. Coherently, using both mature protein database and signal peptidome database could be a promising option to identify some missing proteins by targeting their unique N-terminal tryptic peptide from mature protein database and or C-terminus tryptic peptide from signal peptidome database. In conclusion, Identification of missing protein requires additional consideration during sample preparation, extraction, digestion and data analysis to increase its incidence of identification. Copyright © 2016. Published by Elsevier B.V.
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Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P
2013-06-10
We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc. All rights reserved.
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Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.
Houimel, Mehdi; Mazzucchelli, Luca
2013-01-01
Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Copyright © 2012 Elsevier B.V. All rights reserved.
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Wang, Cong; Tu, Maolin; Wu, Di; Chen, Hui; Chen, Cheng; Wang, Zhenyu; Jiang, Lianzhou
2018-04-11
In the present study, a novel angiotensin I-converting enzyme inhibitory (ACE inhibitory) peptide, EPNGLLLPQY, derived from walnut seed storage protein, fragment residues 80-89, was identified by ultra-high performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) from walnut protein hydrolysate. The IC 50 value of the peptide was 233.178 μM, which was determined by the high performance liquid chromatography method by measuring the amount of hippuric acid (HA) generated from the ACE decomposition substrate (hippuryl-l-histidyl-l-leucine (HHL) to assess the ACE activity. Enzyme inhibitory kinetics of the peptide against ACE were also conducted, by which the inhibitory mechanism of ACE-inhibitory peptide was confirmed. Moreover, molecular docking was simulated by Discovery Studio 2017 R2 software to provide the potential mechanisms underlying the ACE-inhibitory activity of EPNGLLLPQY.
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Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups.
Haaf, Erik; Schlosser, Andreas
2012-01-03
We have developed a new method that applies acidic catalysis with hydrochloric acid for (18)O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95-97 atom % incorporation of (18)O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides. © 2011 American Chemical Society
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SATPdb: a database of structurally annotated therapeutic peptides
Singh, Sandeep; Chaudhary, Kumardeep; Dhanda, Sandeep Kumar; Bhalla, Sherry; Usmani, Salman Sadullah; Gautam, Ankur; Tuknait, Abhishek; Agrawal, Piyush; Mathur, Deepika; Raghava, Gajendra P.S.
2016-01-01
SATPdb (http://crdd.osdd.net/raghava/satpdb/) is a database of structurally annotated therapeutic peptides, curated from 22 public domain peptide databases/datasets including 9 of our own. The current version holds 19192 unique experimentally validated therapeutic peptide sequences having length between 2 and 50 amino acids. It covers peptides having natural, non-natural and modified residues. These peptides were systematically grouped into 10 categories based on their major function or therapeutic property like 1099 anticancer, 10585 antimicrobial, 1642 drug delivery and 1698 antihypertensive peptides. We assigned or annotated structure of these therapeutic peptides using structural databases (Protein Data Bank) and state-of-the-art structure prediction methods like I-TASSER, HHsearch and PEPstrMOD. In addition, SATPdb facilitates users in performing various tasks that include: (i) structure and sequence similarity search, (ii) peptide browsing based on their function and properties, (iii) identification of moonlighting peptides and (iv) searching of peptides having desired structure and therapeutic activities. We hope this database will be useful for researchers working in the field of peptide-based therapeutics. PMID:26527728
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Prandi, Barbara; Farioli, Laura; Tedeschi, Tullia; Pastorello, Elide Anna; Sforza, Stefano
2012-12-30
Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology. Copyright © 2012 John Wiley & Sons, Ltd.
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Common Data Analysis Pipeline | Office of Cancer Clinical Proteomics Research
CPTAC supports analyses of the mass spectrometry raw data (mapping of spectra to peptide sequences and protein identification) for the public using a Common Data Analysis Pipeline (CDAP). The data types available on the public portal are described below. A general overview of this pipeline can be downloaded here. Mass Spectrometry Data Formats RAW (Vendor) Format
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Department of Clinical Investigation: Annual Research Progress Report for Fiscal Year 1992. Volume 2
1993-01-01
graded hypotension (MAP lowered in 10 mm Hg increments range 90 mm - 30 mm Hg), and hypothermia to 30 C. To study the effects of the commonly used inhaled ...peptide-poly-L-lysine conjugates were added to trehalose dimycolate and monophosphoryl lipid A (immune adjuvants) in oil-in-water micelles to aid in
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Ebner, Jennifer; Aşçı Arslan, Ayşe; Fedorova, Maria; Hoffmann, Ralf; Küçükçetin, Ahmet; Pischetsrieder, Monika
2015-03-18
Kefir has a long tradition in human nutrition due to its presupposed health promoting effects. To investigate the potential contribution of bioactive peptides to the physiological effects of kefir, comprehensive analysis of the peptide profile was performed by nano-ESI-LTQ-Orbitrap MS coupled to nano-ultrahigh-performance liquid chromatography. Thus, 257 peptides were identified, mainly released from β-casein, followed by αS1-, κ-, and αS2-casein. Most (236) peptides were uniquely detected in kefir, but not in raw milk indicating that the fermentation step does not only increase the proteolytic activity 1.7- to 2.4-fold compared to unfermented milk, but also alters the composition of the peptide fraction. The influence of the microflora was determined by analyzing kefir produced from traditional kefir grains or commercial starter culture. Kefir from starter culture featured 230 peptide sequences and showed a significantly, 1.4-fold higher proteolytic activity than kefir from kefir grains with 127 peptides. A match of 97 peptides in both varieties indicates the presence of a typical kefir peptide profile that is not influenced by the individual composition of the microflora. Sixteen of the newly identified peptides were previously described as bioactive, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, immunomodulating, opioid, mineral binding, antioxidant, and antithrombotic effects. The present study describes a comprehensive peptide profile of kefir comprising 257 sequences. The peptide list was used to identify 16 bioactive peptides with ACE-inhibitory, antioxidant, antithrombotic, mineral binding, antimicrobial, immunomodulating and opioid activity in kefir. Furthermore, it was shown that a majority of the kefir peptides were not endogenously present in the raw material milk, but were released from milk caseins by proteases of the microbiota and are therefore specific for the product. Consequently, the proteolytic activity and the composition of the peptide profile can be controlled by the applied microflora (grains or starter culture). On the other hand, a considerable portion of the peptide profile was identified to be typical for kefir in general and independent from production parameters. In summary, the generated kefir peptide profile helped to reveal its origin and to identify bioactive peptides in kefir, which may advance the understanding of health benefits of this food product. The results further indicate that subsets of the kefir peptide list can be used as markers to control food authenticity, for example, to distinguish different types of kefir. Copyright © 2015 Elsevier B.V. All rights reserved.
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The Assay Development Working Group (ADWG) of the CPTAC Program is currently drafting a document to propose best practices for generation, quantification, storage, and handling of peptide standards used for mass spectrometry-based assays, as well as interpretation of quantitative proteomic data based on peptide standards. The ADWG is seeking input from commercial entities that provide peptide standards for mass spectrometry-based assays or that perform amino acid analysis.
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Protein and gene model inference based on statistical modeling in k-partite graphs.
Gerster, Sarah; Qeli, Ermir; Ahrens, Christian H; Bühlmann, Peter
2010-07-06
One of the major goals of proteomics is the comprehensive and accurate description of a proteome. Shotgun proteomics, the method of choice for the analysis of complex protein mixtures, requires that experimentally observed peptides are mapped back to the proteins they were derived from. This process is also known as protein inference. We present Markovian Inference of Proteins and Gene Models (MIPGEM), a statistical model based on clearly stated assumptions to address the problem of protein and gene model inference for shotgun proteomics data. In particular, we are dealing with dependencies among peptides and proteins using a Markovian assumption on k-partite graphs. We are also addressing the problems of shared peptides and ambiguous proteins by scoring the encoding gene models. Empirical results on two control datasets with synthetic mixtures of proteins and on complex protein samples of Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana suggest that the results with MIPGEM are competitive with existing tools for protein inference.
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Christiansen, Anders; Kringelum, Jens V; Hansen, Christian S; Bøgh, Katrine L; Sullivan, Eric; Patel, Jigar; Rigby, Neil M; Eiwegger, Thomas; Szépfalusi, Zsolt; de Masi, Federico; Nielsen, Morten; Lund, Ole; Dufva, Martin
2015-08-06
Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.
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Hemoglobin Brigham (α2Aβ2100 Pro→Leu). HEMOGLOBIN VARIANT ASSOCIATED WITH FAMILIAL ERYTHROCYTOSIS
Lokich, Jacob J.; Moloney, William C.; Bunn, H. Franklin; Bruckheimer, Sally M.; Ranney, Helen M.
1973-01-01
Erythrocytosis associated with the presence of a hemoglobin with increased oxygen affinity has been reported for 10 hemoglobin variants, most of which demonstrate altered electrophoretic mobility. Several members of a family were found to have erythrocytosis, and both the whole blood and the hemoglobin exhibited increased oxygen affinity. Phosphate-free hemoglobin solutions had a normal Bohr effect and reactivity to 2,3-diphosphoglycerate. The electrophoretic properties of the hemoglobin were normal, but on peptide mapping of a tryptic digest of the isolated β-chains, a normal βT11 peptide and an abnormal βT11 with greater Rf were seen. Analysis of the abnormal peptide showed the substitution of leucine for the normal proline at β100 (helical residue G2). The hemoglobin variant, designated Hb Brigham, serves to emphasize the necessity for detailed evaluation of the structure and function of hemoglobin in familial erythrocytosis even with electrophoretically “normal” hemoglobin. PMID:4719677
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The active site of O-GlcNAc transferase imposes constraints on substrate sequence
Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.
2016-01-01
O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509
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Luzar, J; Štrukelj, B; Lunder, M
2016-11-01
Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Serang, Oliver; MacCoss, Michael J.; Noble, William Stafford
2010-01-01
The problem of identifying proteins from a shotgun proteomics experiment has not been definitively solved. Identifying the proteins in a sample requires ranking them, ideally with interpretable scores. In particular, “degenerate” peptides, which map to multiple proteins, have made such a ranking difficult to compute. The problem of computing posterior probabilities for the proteins, which can be interpreted as confidence in a protein’s presence, has been especially daunting. Previous approaches have either ignored the peptide degeneracy problem completely, addressed it by computing a heuristic set of proteins or heuristic posterior probabilities, or by estimating the posterior probabilities with sampling methods. We present a probabilistic model for protein identification in tandem mass spectrometry that recognizes peptide degeneracy. We then introduce graph-transforming algorithms that facilitate efficient computation of protein probabilities, even for large data sets. We evaluate our identification procedure on five different well-characterized data sets and demonstrate our ability to efficiently compute high-quality protein posteriors. PMID:20712337
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Ghorab, Hamida; Lammi, Carmen; Arnoldi, Anna; Kabouche, Zahia; Aiello, Gilda
2018-01-15
An investigation on the proteome of the sweet kernel of apricot, based on equalisation with combinatorial peptide ligand libraries (CPLLs), SDS-PAGE, nLC-ESI-MS/MS, and database search, permitted identifying 175 proteins. Gene ontology analysis indicated that their main molecular functions are in nucleotide binding (20.9%), hydrolase activities (10.6%), kinase activities (7%), and catalytic activity (5.6%). A protein-protein association network analysis using STRING software permitted to build an interactomic map of all detected proteins, characterised by 34 interactions. In order to forecast the potential health benefits deriving from the consumption of these proteins, the two most abundant, i.e. Prunin 1 and 2, were enzymatically digested in silico predicting 10 and 14 peptides, respectively. Searching their sequences in the database BIOPEP, it was possible to suggest a variety of bioactivities, including dipeptidyl peptidase-IV (DPP-IV) and angiotensin converting enzyme I (ACE) inhibition, glucose uptake stimulation and antioxidant properties. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Conformational states of the full-length glucagon receptor
Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang
2015-01-01
Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798
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Pepperl, Sandra; Benninger-Döring, Gerlinde; Modrow, Susanne; Wolf, Hans; Jilg, Wolfgang
1998-01-01
We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells. PMID:9765404
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Improving Peptide Applications Using Nanotechnology.
Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P
2016-01-01
Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.
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Application of Immunosignatures for Diagnosis of Valley Fever
Navalkar, Krupa Arun; Johnston, Stephen Albert; Woodbury, Neal; Galgiani, John N.; Magee, D. Mitchell; Chicacz, Zbigniew
2014-01-01
Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics. PMID:24964807
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Toledo-Machado, Christina Monerat; Bueno, Lilian Lacerda; Menezes-Souza, Daniel; Machado-de-Avila, Ricardo Andrez; Nguyen, Christophe; Granier, Claude; Bartholomeu, Daniella Castanheira; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio
2015-02-28
Leishmania parasites can cause visceral or cutaneous disease and are found in subtropical and tropical regions of the Old and New World. The pathology of the infection is determined by both host immune factors and species/strain differences of the parasite. Dogs represent the major reservoir of Leishmania infantum (syn. L. chagasi) and vaccines are considered the most cost-effective control tools for canine disease. Selection of immunodominant peptides was performed by Phage Display to identify sequences recognized by L. infantum naturally infected animals. Sera from Leishmania infected animals were used in the biopanning to selection of specific peptides. Serum samples from T. cruzi infected and healthy animals were used as control. After selection, synthetic peptides were produced in membrane (spot-synthesis) in soluble form and blotting and ELISA were performed for validation of serum reactivity. Selected peptide was formulated with aluminum hydroxide and liposomes and immunization was performed in BALB/c mice. Protection was determined by qPCR after challenge infection with virulent L. infantum. We reported the selection of Peptide 5 through Phage Display technique and demonstrate its ability to promote a state of immunity against L. infantum infection in murine model after immunization using liposomes as vaccine carrier. Our results demonstrate that immunization with Peptide 5 when formulated with aluminum hydroxide and liposomes is immunogenic and elicited significant protection associated with the induction of mixed Th1/Th2 immune response against L. infantum infection. Peptide 5 is a promising vaccine candidate and the findings obtained in the present study encourage canine trials to confirm the effectiveness of a vaccine against CVL.
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DeepSig: deep learning improves signal peptide detection in proteins.
Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita
2018-05-15
The identification of signal peptides in protein sequences is an important step toward protein localization and function characterization. Here, we present DeepSig, an improved approach for signal peptide detection and cleavage-site prediction based on deep learning methods. Comparative benchmarks performed on an updated independent dataset of proteins show that DeepSig is the current best performing method, scoring better than other available state-of-the-art approaches on both signal peptide detection and precise cleavage-site identification. DeepSig is available as both standalone program and web server at https://deepsig.biocomp.unibo.it. All datasets used in this study can be obtained from the same website. pierluigi.martelli@unibo.it. Supplementary data are available at Bioinformatics online.
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Phage display: concept, innovations, applications and future.
Pande, Jyoti; Szewczyk, Magdalena M; Grover, Ashok K
2010-01-01
Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field. Copyright © 2010 Elsevier Inc. All rights reserved.
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Bulau, Patrick; Okuno, Atsuro; Thome, Elke; Schmitz, Tina; Peter-Katalinic, Jasna; Keller, Rainer
2005-11-01
The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.
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Zhang, Xi
2016-01-01
Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins—at high efficiency and peptide reproducibility—poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. PMID:27073180
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McMurtrey, Curtis P; Lelic, Alina; Piazza, Paolo; Chakrabarti, Ayan K; Yablonsky, Eric J; Wahl, Angela; Bardet, Wilfried; Eckerd, Annette; Cook, Robert L; Hess, Rachael; Buchli, Rico; Loeb, Mark; Rinaldo, Charles R; Bramson, Jonathan; Hildebrand, William H
2008-02-26
Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.
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Wu, Chunxiao; Wang, Shu
2012-01-01
Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.
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Dolimbek, Behzod Z; Steward, Lance E; Aoki, K Roger; Atassi, M Zouhair
2008-02-01
The purpose of this work was to map the continuous regions recognized by human, horse and mouse anti-botulinum neurotoxin B (BoNT/B) antibodies (Abs). We synthesized a panel of sixty 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and together encompassed the entire heavy chain of BoNT/B (H/B, residues 442-1291). Abs from the three host species recognized similar, but not identical, peptides. There were also peptides recognized by two or only by one host species. Where a peptide was recognized by Abs of more than one host species, these Abs were at different levels among the species. Human, horse and mouse Abs bound, although in different amounts, to regions within peptides 736-754, 778-796, 848-866, 932-950, 974-992, 1058-1076 and 1128-1146. Human and horse Abs bound to peptides 890-908 and 1170-1188. Human and mouse Abs recognized peptides 470-488/484-502 overlap, 638-656, 722-740, 862-880, 1030-1048, 1072-1090, 1240-1258 and 1268-1291. We concluded that the antigenic regions localized with the three antisera are quite similar, exhibiting in some cases a small shift to the left or to the right. This is consistent with what is known about protein immune recognition. In the three-dimensional structure, the regions recognized on H/B by anti-BoNT/B Abs occupied surface locations and analysis revealed no correlation between these surface locations and surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. A region that bound mouse Abs overlapped with a recently defined site on BoNT/B that binds to mouse and rat synaptotagmin II, thus providing a molecular explanation for the blocking (protecting) activity of these Abs. The regions thus localized afford candidates for incorporation into a synthetic vaccine design.
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Atassi, M Zouhair; Dolimbek, Behzod Z; Jankovic, Joseph; Steward, Lance E; Aoki, K Roger
2011-07-01
This work was aimed at determining the BoNT/A L-chain antigenic regions recognized by blocking antibodies in human antisera from cervical dystonia patients who had become immunoresistant to BoNT/A treatment. Antisera from 28 immunoresistant patients were analyzed for binding to each of 32 overlapping synthetic peptides that spanned the entire L-chain. A mixture of the antisera showed that antibodies bound to three peptides, L11 (residues 141-159), L14 (183-201) and L18 (239-257). When mapped separately, the antibodies were bound only by a limited set of peptides. No peptide bound antibodies from all the patients and amounts of antibodies bound to a given peptide varied with the patient. Peptides L11, L14 and L18 were recognized predominantly. A small but significant number of patients had antibodies to peptides L27 (365-383) and L29 (379-397). Other peptides were recognized at very low and perhaps insignificant antibody levels by a minority (15% or less) of patients or had no detectable antibody with any of the sera. In the 3-dimensional structure, antibody-binding regions L11, L14 and L18 of the L-chain occupy surface areas and did not correlate with electrostatic potential, hydrophilicity/hydrophobicity, or temperature factor. These three antigenic regions reside in close proximity to the belt of the heavy chain. The regions L11 and L18 are accessible in both the free light chain and the holotoxin forms, while L14 appears to be less accessible in the holotoxin. Antibodies against these regions could prevent delivery of the L-chain into the neurons by inhibition of the translocation. Copyright © 2011 Elsevier GmbH. All rights reserved.
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Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.
Lluch-Senar, Maria; Mancuso, Francesco M; Climente-González, Héctor; Peña-Paz, Marcia I; Sabido, Eduard; Serrano, Luis
2016-02-01
A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33,413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe-Tyr and Ala-Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or had Arg/Lys at the C-terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51,453 out of the 70,040 initial acquired spectra). All MS data have been deposited in the ProteomeXchange with identifier PXD002779 (http://proteomecentral.proteomexchange.org/dataset/PXD002779). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Peptide/laccase cocatalyzed asymmetric α-oxyamination of aldehydes.
Akagawa, Kengo; Kudo, Kazuaki
2011-07-01
An asymmetric α-oxyamination could be successfully performed by a peptide catalyst and laccase. The combination of peptide catalysis and enzymatic air oxidation promoted the reaction smoothly in water without employing a metal reagent. The oxyaminated compounds could be obtained as both aldehyde and carboxylic acid products depending on the reaction conditions.
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Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M
1999-03-01
Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.
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Deng, Youping; Xu, Hu; Riedel, Heimo
2007-02-15
The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.
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Mooney, Catherine; Haslam, Niall J.; Pollastri, Gianluca; Shields, Denis C.
2012-01-01
The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides. We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4–20 amino acids) and one focused on long peptides ( amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure. We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive. PMID:23056189
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Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties
Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.
2004-01-01
A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854
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Hilpert, Kai; Winkler, Dirk F H; Hancock, Robert E W
2007-01-01
Peptide synthesis on cellulose using SPOT technology allows the parallel synthesis of large numbers of addressable peptides in small amounts. In addition, the cost per peptide is less than 1% of peptides synthesized conventionally on resin. The SPOT method follows standard fluorenyl-methoxy-carbonyl chemistry on conventional cellulose sheets, and can utilize more than 600 different building blocks. The procedure involves three phases: preparation of the cellulose membrane, stepwise coupling of the amino acids and cleavage of the side-chain protection groups. If necessary, peptides can be cleaved from the membrane for assays performed using soluble peptides. These features make this method an excellent tool for screening large numbers of peptides for many different purposes. Potential applications range from simple binding assays, to more sophisticated enzyme assays and studies with living microbes or cells. The time required to complete the protocol depends on the number and length of the peptides. For example, 400 9-mer peptides can be synthesized within 6 days.
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Analysis of iodinated quorum sensing peptides by LC-UV/ESI ion trap mass spectrometry.
Janssens, Yorick; Verbeke, Frederick; Debunne, Nathan; Wynendaele, Evelien; Peremans, Kathelijne; De Spiegeleer, Bart
2018-02-01
Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C 18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%).
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Synthesis and Primary Characterization of Self-Assembled Peptide-Based Hydrogels
Nagarkar, Radhika P.; Schneider, Joel P.
2009-01-01
Summary Hydrogels based on peptide self-assembly form an important class of biomaterials that find application in tissue engineering and drug delivery. It is essential to prepare peptides with high purity to achieve batch-to-batch consistency affording hydrogels with reproducible properties. Automated solid-phase peptide synthesis coupled with optimized Fmoc (9-fluorenylmethoxycarbonyl) chemistry to obtain peptides in high yield and purity is discussed. Details of isolating a desired peptide from crude synthetic mixtures and assessment of the peptide’s final purity by high-performance liquid chromatography and mass spectrometry are provided. Beyond the practical importance of synthesis and primary characterization, techniques used to investigate the properties of hydrogels are briefly discussed. PMID:19031061
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Luo, Heng; Ye, Hao; Ng, Hui; Shi, Leming; Tong, Weida; Mattes, William; Mendrick, Donna; Hong, Huixiao
2015-01-01
As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Network analysis is a useful approach for analyzing large and sparse datasets such as the HLA-peptide binding dataset. The modules identified from the network analysis clustered peptides and HLAs with similar sequences and properties of amino acids. Nebula performed well in the predictions of HLA-peptide binding. We demonstrated that network analysis coupled with Nebula is an efficient approach to understand and predict HLA-peptide binding interactions and thus, could further our understanding of ADRs.
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2015-01-01
Background As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Methods Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Results Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Conclusions Network analysis is a useful approach for analyzing large and sparse datasets such as the HLA-peptide binding dataset. The modules identified from the network analysis clustered peptides and HLAs with similar sequences and properties of amino acids. Nebula performed well in the predictions of HLA-peptide binding. We demonstrated that network analysis coupled with Nebula is an efficient approach to understand and predict HLA-peptide binding interactions and thus, could further our understanding of ADRs. PMID:26424483
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Analysis of illegal peptide drugs via HILIC-DAD-MS.
Janvier, Steven; De Sutter, Evelien; Wynendaele, Evelien; De Spiegeleer, Bart; Vanhee, Celine; Deconinck, Eric
2017-11-01
Biopharmaceuticals have established themselves as highly efficient medicines, and are still one of the fastest growing parts of the health-product industry. Unfortunately, the introduction of these promising new drugs went hand in hand with the creation of a black market for illegal and counterfeit biotechnology drugs. Particularly popular are the lyophilised peptides with a molecular weight of less than 5kDa. Most of them are meant for subcutaneous injection and are easily accessible via the internet. In recent years, different methods based on reversed phase liquid chromatography have been developed to detect and quantify these peptides. The emerging of more polar peptides however requires the introduction of other separation techniques. Therefore, we set out to develop and validate an analytical method based on hydrophilic interaction liquid chromatography (HILIC) to identify and quantify the most frequently encountered illegal peptides on the European market. For this objective, five different HILIC columns were selected and screened for their chromatographic performance. Among those columns, the ZIC HILIC column showed the best performance under the tested screening conditions in terms of resolution and symmetry factor for the targeted peptide set. Hence, the operational conditions were further optimised for the identification of illegal preparations via mass spectrometry (MS) and quantification via UV. Validation was performed via accuracy profiles based on the ISO 17025 guideline. The obtained validated HILIC-method allows for the detection and quantification of the most frequently encountered illegal peptides on the internet in a total run time of 35min including post gradient equilibration and online cleaning step. Combined with a previously developed RPLC-method, the ZIC HILIC system allows for the detection and quantification of a wide spectrum of illicit peptide drugs available on the internet. Furthermore, the developed method could also be envisaged for the detection of new emerging polar peptide drugs. Copyright © 2017 Elsevier B.V. All rights reserved.
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Tomabechi, Yusuke; Katoh, Toshihiko; Kunishima, Munetaka; Inazu, Toshiyuki; Yamamoto, Kenji
2017-08-01
For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.
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Hou, Tingjun; Zhang, Wei; Case, David A; Wang, Wei
2008-02-29
Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was used to assess the performances of different kernel functions in SVM and different combinations of the MIECs. The best SVM classifier gave satisfactory predictions for the test set, indicated by average prediction accuracy rates of 78% and 91% for the binding and non-binding peptides, respectively. We also showed that the performance of our approach on both binding affinity prediction and binder/non-binder classification was superior to the performances of the conventional MM/Poisson-Boltzmann solvent-accessible surface area and MM/generalized Born solvent-accessible surface area calculations. Our study demonstrates that the analysis of the MIECs between peptides and the SH3 domain can successfully characterize the binding interface, and it provides a framework to derive integrated prediction models for different domain-peptide systems.
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Ronn, Jonas; Jensen, Elisa P; Wewer Albrechtsen, Nicolai J; Holst, Jens Juul; Sorensen, Charlotte M
2017-12-01
Glucagon-like peptide-1 (GLP-1) is an incretin hormone increasing postprandial insulin release. GLP-1 also induces diuresis and natriuresis in humans and rodents. The GLP-1 receptor is extensively expressed in the renal vascular tree in normotensive rats where acute GLP-1 treatment leads to increased mean arterial pressure (MAP) and increased renal blood flow (RBF). In hypertensive animal models, GLP-1 has been reported both to increase and decrease MAP. The aim of this study was to examine expression of renal GLP-1 receptors in spontaneously hypertensive rats (SHR) and to assess the effect of acute intrarenal infusion of GLP-1. We hypothesized that GLP-1 would increase diuresis and natriuresis and reduce MAP in SHR. Immunohistochemical staining and in situ hybridization for the GLP-1 receptor were used to localize GLP-1 receptors in the kidney. Sevoflurane-anesthetized normotensive Sprague-Dawley rats and SHR received a 20 min intrarenal infusion of GLP-1 and changes in MAP, RBF, heart rate, dieresis, and natriuresis were measured. The vasodilatory effect of GLP-1 was assessed in isolated interlobar arteries from normo- and hypertensive rats. We found no expression of GLP-1 receptors in the kidney from SHR. However, acute intrarenal infusion of GLP-1 increased MAP, RBF, dieresis, and natriuresis without affecting heart rate in both rat strains. These results suggest that the acute renal effects of GLP-1 in SHR are caused either by extrarenal GLP-1 receptors activating other mechanisms (e.g., insulin) to induce the renal changes observed or possibly by an alternative renal GLP-1 receptor. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
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Experimental conformational energy maps of proteins and peptides.
Balaji, Govardhan A; Nagendra, H G; Balaji, Vitukudi N; Rao, Shashidhar N
2017-06-01
We have presented an extensive analysis of the peptide backbone dihedral angles in the PDB structures and computed experimental Ramachandran plots for their distributions seen under a various constraints on X-ray resolution, representativeness at different sequence identity percentages, and hydrogen bonding distances. These experimental distributions have been converted into isoenergy contour plots using the approach employed previously by F. M. Pohl. This has led to the identification of energetically favored minima in the Ramachandran (ϕ, ψ) plots in which global minima are predominantly observed either in the right-handed α-helical or the polyproline II regions. Further, we have identified low energy pathways for transitions between various minima in the (ϕ,ψ) plots. We have compared and presented the experimental plots with published theoretical plots obtained from both molecular mechanics and quantum mechanical approaches. In addition, we have developed and employed a root mean square deviation (RMSD) metric for isoenergy contours in various ranges, as a measure (in kcal.mol -1 ) to compare any two plots and determine the extent of correlation and similarity between their isoenergy contours. In general, we observe a greater degree of compatibility with experimental plots for energy maps obtained from molecular mechanics methods compared to most quantum mechanical methods. The experimental energy plots we have investigated could be helpful in refining protein structures obtained from X-ray, NMR, and electron microscopy and in refining force field parameters to enable simulations of peptide and protein structures that have higher degree of consistency with experiments. Proteins 2017; 85:979-1001. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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2015-01-01
High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200
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Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G
2014-04-04
High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.
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Yang, Huirong; Zong, Xuyan; Cui, Chun; Mu, Lixia; Zhao, Haifeng
2017-12-22
Lys and Leu were generally considered as the key amino acids for brewer's yeast during beer brewing. In the present study, peptide Lys-Leu and a free amino acid (FAA) mixture of Lys and Leu (Lys + Leu) were supplemented in 24 °P wort to examine their effects on physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation. Results showed that although both peptide Lys-Leu and their FAA mixture supplementations could increase the growth and viability, intracellular trehalose and glycerol content, wort fermentability, and ethanol content for brewer's yeast during VHG wort fermentation, and peptide was better than their FAA mixture at promoting growth and fermentation for brewer's yeast when the same dose was kept. Moreover, peptide Lys-Leu supplementation significantly increased the assimilation of Asp, but decreased the assimilation of Gly, Ala, Val, (Cys)2, Ile, Leu, Tyr, Phe, Lys, Arg, and Pro. However, the FAA mixture supplementation only promoted the assimilation of Lys and Leu, while reduced the absorption of total amino acids to a greater extent. Thus, the peptide Lys-Leu was more effective than their FAA mixture on the improvement of physiological activity, fermentation performance, and nitrogen metabolism of brewer's yeast during VHG wort fermentation. © 2017 International Union of Biochemistry and Molecular Biology, Inc.
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An unusual form of lipid linkage to the CD45 peptide
DOE Office of Scientific and Technical Information (OSTI.GOV)
Takeda, Akiko; Maizel, A.L.
1990-11-02
Some protein kinases and phosphatases are myristoylated on their amino terminus, which perhaps contributes to subcellular localization or regulation. Glycoprotein CD45, a hematopoietic tyrosine phosphatase, was examined for fatty acid content. The CD45 protein incorporated ({sup 3}H)myristate, but little ({sup 3}H)palmitate. The label was not metabolized and reincorporated into amino acids or saccharides, as revealed by peptide maps of CD45 labeled with ({sup 3}H)myristate, {sup 14}C-labeled amino acids, ({sup 35}S)methionine, or {sup 125}I, and glycosidase treatments, respectively. The myristate label was resistant to mild alkaline methanolysis and was found in fatty acid and sphingosine, indicating an unusual form of lipidmore » attachment to CD45.« less
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Zhao, Lei; Whiteaker, Jeffrey R; Voytovich, Uliana J; Ivey, Richard G; Paulovich, Amanda G
2015-10-02
Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring mass spectrometry (immuno-MRM) enables precise quantification of peptides. Affinity-purified polyclonal antibodies are routinely used as affinity reagents in immuno-MRM assays, but they are not renewable, limiting the number of experiments that can be performed. In this technical note, we describe a workflow to regenerate anti-peptide polyclonal antibodies coupled to magnetic beads for enrichments in multiplex immuno-MRM assays. A multiplexed panel of 44 antibodies (targeting 60 peptides) is used to show that peptide analytes can be effectively stripped off of antibodies using acid washing without compromising assay performance. The performance of the multiplexed panel (determined by correlation, agreement, and precision of reused assays) is reproducible (R(2) between 0.81 and 0.99) and consistent (median CVs 8-15%) for at least 10 times of washing and reuse. Application of this workflow to immuno-MRM studies greatly reduces per sample assay cost and increases the number of samples that can be interrogated with a limited supply of polyclonal antibody reagent. This allows more characterization for promising and desirable targets prior to committing funds and efforts to conversion to a renewable monoclonal antibody.
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Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs.
Ramaker, Katrin; Henkel, Maik; Krause, Thorsten; Röckendorf, Niels; Frey, Andreas
2018-11-01
Delivering reagents into cells is a key demand in molecular medicine. The vehicle of choice is often cell penetrating peptides (CPPs), which can ferry conjugated cargo across membranes. Although numerous peptides have been shown to promote such uptake events, there has been no comprehensive comparison of individual performance under standardized conditions. We have devised a method to rapidly analyze the ability of a multitude of CPP conjugates to carry a model cargo into HeLa cells. Sequence information for 474 CPPs was collected from literature sources, and the respective peptides were synthesized and modified with carboxyfluorescein (FAM) as model cargo. All candidates were evaluated in an identical uptake test, and transport was quantified using cellular fluorescence intensities. Substantial differences in the ability to carry the fluorophore into the cells were observed, with transport performance differing by a factor of 70 between the best CPP investigated and cargo alone. Strong correlations were observed between uptake efficiency and both sequence length and the presence of positive net charge. A compilation of the 20 top performers with regard to cargo delivery performance and cell compatibility is provided.
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99mTc-D(LPR): A novel retro-inverso peptide for VEGF receptor-1 targeted tumor imaging.
Rezazadeh, Farzaneh; Sadeghzadeh, Nourollah; Abedi, Seyed Mohammad; Abediankenari, Saeid
2018-05-31
The aim of this study was to evaluate the ability of D (LPR), a novel retro-inverso peptidomimetic derivative for imaging colon cancer. Two different D (LPR) analogs were designed and compared based on conjugation of HYNIC at peptide's C or N terminal and then labeled with technetium-99m using tricine/EDDA as an exchange coligands. The radiolabeled conjugates were assessed for in vitro stability in saline and serum. The VEGFR-1 and NRP-1 receptors affinity, in vitro internalization and also dissociation Constance was evaluated. SPCET imaging and biodistribution studies were performed in nude mice bearing HT-29 xenograft tumors. Both peptides labeled with technetium-99m in high radiochemical yield (˃97%). Peptide stability studies indicated a high metabolic stability of the radiopeptides in solution and serum. In vitro blocking studies demonstrated specific binding and internalization of [ 99m Tc]Tc-HYNIC-peptides in cultured HUVEC cells. The K d value for 99m Tc-peptide 1 and 99m Tc-peptide 2 were found to be 56.8 ± 12.9 nM and 71.6 ± 17.9 nM respectively. The tumor to muscle ratio was significant at 0.5 and 1 h after injection (4.5 and 4 for 99m Tc-peptide 1 and 4.9 and 4.4 for 99m Tc-peptide 2 at 0.5 and 1 h p.i. respectively). SPECT imaging studies revealed that both radioconjugates had prominent activity accumulation in VEGFR-1 and NRP-1 expressing HT-29 tumors. This study is the first instance of using a radiolabeled retro-inverso peptide for tumor imaging which is a promising tool to improve the performance of fragile peptide probes in vivo as imaging agents and warrant further investigations in other peptide-target systems. Copyright © 2018 Elsevier Inc. All rights reserved.
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C-peptide does not affect ocular blood flow in patients with type 1 diabetes.
Polska, Elzbieta; Kolodjaschna, Julia; Berisha, Fatmire; Malec, Maria M; Simader, Christian; Bayerle-Eder, Michaela; Roden, Michael; Schmetterer, Leopold
2006-09-01
The aim of the present study was to investigate the effect of intravenous C-peptide infusion on ocular blood flow in patients with type 1 diabetes under euglycemic conditions. The study was performed in a randomized, placebo-controlled, double-masked, two-way, crossover design in 10 type 1 diabetic patients. C-peptide was intravenously administered at two different dosages (dosage 1: 25 pmol . kg(-1) . min(-1) bolus followed by 5 pmol . kg(-1) . min(-1) continuous infusion; dosage 2: six times higher than dosage 1), each for 60 min. Physiologic saline solution was used as a control for C-peptide on a different study day. On both study days, euglycemic clamps were performed. To assess retinal blood flow, laser Doppler velocimetry (blood flow velocities) and retinal vessel analyzer (vessels diameters) measurements were performed. Laser interferometric measurements of fundus pulsation were used to assess pulsatile choroidal blood flow. Blood velocities in the ophthalmic artery were measured using color Doppler imaging. Eight patients (two female and six male) completed the study according to the protocol and without adverse events. One patient developed an anaphylactic reaction to C-peptide, which resolved without sequelae. The following results originate from the remaining eight subjects. Systemic hemodynamic parameters remained stable during both study days. Infusion of C-peptide did not affect any ocular hemodynamic parameter. The data of the present study indicate that exogenous C-peptide exerts no effect on ocular hemodynamic parameters in type 1 diabetic patients under euglycemic conditions. The maximum detectable change in these parameters was <25%.
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Ma, Zhi-Li; Hou, Tao; Shi, Wen; Liu, Wei-Wei; Ibrahim, Salam A; He, Hui
2016-11-01
In this study, peptides that facilitate alcohol metabolism were purified and identified from corn protein hydrolysates. The ultra-filtered fraction with a molecular weight < 3 kDa (F3) potential activity was separated into six fractions (F3-H1-F3-H6) by semi-preparative high-performance liquid chromatography. Among the resultant six fractions, F3-H4 and F3-H5 exhibited the highest ability to eliminate alcohol in vivo. A total of 16 peptides with strong signal values were identified from F3-H4 and F3-H5 fractions by nano liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Several identified peptides were then selected and synthesized to determine their potential to facilitate alcohol metabolism. We found that Leu-Leu and Pro-Phe were the key structure units in Gln-Leu-Leu-Pro-Phe responsible for this peptide's ability to facilitate alcohol metabolism. However, the role of Leu-Leu and Pro-Phe may be affected by peptide chain length and hydrophobic properties. Our results have thus provided some insight into the study of the structure-activity relationships of corn peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Degradation of milk-based bioactive peptides by yogurt fermentation bacteria.
Paul, M; Somkuti, G A
2009-09-01
To analyse the effect of cell-associated peptidases in yogurt starter culture strains Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST) on milk-protein-based antimicrobial and hypotensive peptides in order to determine their survival in yogurt-type dairy foods. The 11mer antimicrobial and 12mer hypotensive milk-protein-derived peptides were incubated with mid-log cells of LB and ST, which are required for yogurt production. Incubations were performed at pH 4.5 and 7.0, and samples removed at various time points were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The peptides remained mostly intact at pH 4.5 in the presence of ST strains and moderately digested by exposure to LB cells. Peptide loss occurred more rapidly and was more extensive after incubation at pH 7.0. The 11mer and 12mer bioactive peptides may be added at the end of the yogurt-making process when the pH level has dropped to 4.5, limiting the overall extent of proteolysis. The results show the feasibility of using milk-protein-based antimicrobial and hypotensive peptides as food supplements to improve the health-promoting qualities of liquid and semi-solid dairy foods prepared by the yogurt fermentation process.
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Fine mapping of canine parvovirus B cell epitopes.
López de Turiso, J A; Cortés, E; Ranz, A; García, J; Sanz, A; Vela, C; Casal, J I
1991-10-01
In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.
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Moyer, Bryan D; Murray, Justin K; Ligutti, Joseph; Andrews, Kristin; Favreau, Philippe; Jordan, John B; Lee, Josie H; Liu, Dong; Long, Jason; Sham, Kelvin; Shi, Licheng; Stöcklin, Reto; Wu, Bin; Yin, Ruoyuan; Yu, Violeta; Zou, Anruo; Biswas, Kaustav; Miranda, Les P
2018-01-01
Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.
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Murray, Justin K.; Ligutti, Joseph; Andrews, Kristin; Favreau, Philippe; Jordan, John B.; Lee, Josie H.; Liu, Dong; Long, Jason; Sham, Kelvin; Shi, Licheng; Stöcklin, Reto; Wu, Bin; Yin, Ruoyuan; Yu, Violeta; Zou, Anruo; Biswas, Kaustav; Miranda, Les P.
2018-01-01
Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers. PMID:29723257
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Data Portal | Office of Cancer Clinical Proteomics Research
The CPTAC Data Portal is a centralized repository for the public dissemination of proteomic sequence datasets collected by CPTAC, along with corresponding genomic sequence datasets. In addition, available are analyses of CPTAC's raw mass spectrometry-based data files (mapping of spectra to peptide sequences and protein identification) by individual investigators from CPTAC and by a Common Data Analysis Pipeline.
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HPEPDOCK: a web server for blind peptide-protein docking based on a hierarchical algorithm.
Zhou, Pei; Jin, Bowen; Li, Hao; Huang, Sheng-You
2018-05-09
Protein-peptide interactions are crucial in many cellular functions. Therefore, determining the structure of protein-peptide complexes is important for understanding the molecular mechanism of related biological processes and developing peptide drugs. HPEPDOCK is a novel web server for blind protein-peptide docking through a hierarchical algorithm. Instead of running lengthy simulations to refine peptide conformations, HPEPDOCK considers the peptide flexibility through an ensemble of peptide conformations generated by our MODPEP program. For blind global peptide docking, HPEPDOCK obtained a success rate of 33.3% in binding mode prediction on a benchmark of 57 unbound cases when the top 10 models were considered, compared to 21.1% for pepATTRACT server. HPEPDOCK also performed well in docking against homology models and obtained a success rate of 29.8% within top 10 predictions. For local peptide docking, HPEPDOCK achieved a high success rate of 72.6% on a benchmark of 62 unbound cases within top 10 predictions, compared to 45.2% for HADDOCK peptide protocol. Our HPEPDOCK server is computationally efficient and consumed an average of 29.8 mins for a global peptide docking job and 14.2 mins for a local peptide docking job. The HPEPDOCK web server is available at http://huanglab.phys.hust.edu.cn/hpepdock/.
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Lundegaard, Claus; Lund, Ole; Nielsen, Morten
2008-06-01
Several accurate prediction systems have been developed for prediction of class I major histocompatibility complex (MHC):peptide binding. Most of these are trained on binding affinity data of primarily 9mer peptides. Here, we show how prediction methods trained on 9mer data can be used for accurate binding affinity prediction of peptides of length 8, 10 and 11. The method gives the opportunity to predict peptides with a different length than nine for MHC alleles where no such peptides have been measured. As validation, the performance of this approach is compared to predictors trained on peptides of the peptide length in question. In this validation, the approximation method has an accuracy that is comparable to or better than methods trained on a peptide length identical to the predicted peptides. The algorithm has been implemented in the web-accessible servers NetMHC-3.0: http://www.cbs.dtu.dk/services/NetMHC-3.0, and NetMHCpan-1.1: http://www.cbs.dtu.dk/services/NetMHCpan-1.1
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Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.
Dolashka, Pavlina; Moshtanska, Vesela; Borisova, Valika; Dolashki, Aleksander; Stevanovic, Stefan; Dimanov, Tzvetan; Voelter, Wolfgang
2011-07-01
Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. Copyright © 2011 Elsevier Inc. All rights reserved.
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Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer.
Grant, Irene R; Foddai, Antonio C G; Tarrant, James C; Kunkel, Brenna; Hartmann, Faye A; McGuirk, Sheila; Hansen, Chungyi; Talaat, Adel M; Collins, Michael T
2017-12-01
When advising farmers on how to control Johne's disease in an infected herd, one of the main recommendations is to avoid feeding waste milk to calves and instead feed calf milk replacer (CMR). This advice is based on the assumption that CMR is free of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells, an assumption that has not previously been challenged. We tested commercial CMR products (n = 83) obtained from dairy farms around the United States by the peptide-mediated magnetic separation (PMS)-phage assay, PMS followed by liquid culture (PMS-culture), and direct IS900 quantitative PCR (qPCR). Conventional microbiological analyses for total mesophilic bacterial counts, coliforms, Salmonella, coagulase-negative staphylococci, streptococci, nonhemolytic Corynebacterium spp., and Bacillus spp. were also performed to assess the overall microbiological quality of the CMR. Twenty-six (31.3%) of the 83 CMR samples showed evidence of the presence of MAP. Seventeen (20.5%) tested positive for viable MAP by the PMS-phage assay, with plaque counts ranging from 6 to 1,212 pfu/50 mL of reconstituted CMR (average 248.5 pfu/50 mL). Twelve (14.5%) CMR samples tested positive for viable MAP by PMS-culture; isolates from all 12 of these samples were subsequently confirmed by whole-genome sequencing to be different cattle strains of MAP. Seven (8.4%) CMR samples tested positive for MAP DNA by IS900 qPCR. Four CMR samples tested positive by both PMS-based tests and 5 CMR samples tested positive by IS900 qPCR plus one or other of the PMS-based tests, but only one CMR sample tested positive by all 3 MAP detection tests applied. All conventional microbiology results were within current standards for whole milk powders. A significant association existed between higher total bacterial counts and presence of viable MAP indicated by either of the PMS-based assays. This represents the first published report of the isolation of viable MAP from CMR. Our findings raise concerns about the potential ability of MAP to survive manufacture of dried milk-based products. The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
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Musharraf, Syed Ghulam; Ameer, Mariam; Ali, Arslan
2017-01-05
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization technique, has become a method of choice for high-throughput analysis of proteins and peptides. In this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were employed as target analytes to check performance of olanzapine and compared with conventional MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA. Computational studies were performed to determine the thermochemical properties of olanzapine in order to further evaluate its similarity to MALDI matrices which were found in good agreement with the data of existing MALDI matrices. Copyright © 2016. Published by Elsevier B.V.
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Development of bacterial display peptides for use in biosensing applications
NASA Astrophysics Data System (ADS)
Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.
2012-06-01
Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.
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Joly, Laure; Antoine, Rodolphe; Broyer, Michel; Lemoine, Jérôme; Dugourd, Philippe
2008-02-07
Electron detachment from peptide dianions is studied as a function of the laser wavelength. The first step for the detachment is a resonant electronic excitation of the dianions. Electronic excitation spectra are reported for three peptides, including gramicidin. A comparative study of the detachment yield for 13 peptides was performed at 260 nm and at 220 nm. At 260 nm, the detachment yield is mainly driven by the sum of the absorption coefficients of the aromatic amino acids that are contained in the peptide. At 220 nm, no direct relation is observed between the electron photodetachement yields and the sum of absorption efficiencies. At this wavelength, the sequence and the structure of the peptide may have an influence on the photodetachment process.
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Antimalarial Activities of Peptide Antibiotics Isolated from Fungi
Nagaraj, G.; Uma, M. V.; Shivayogi, M. S.; Balaram, Hemalatha
2001-01-01
Malaria caused by Plasmodium falciparum is a major public health problem in the developing countries of the world. Clinical treatment of malaria has become complicated due to the occurrence of infections caused by drug resistant parasites. Secondary metabolites from fungi are an attractive source of chemotherapeutic agents. This work reports the isolation and in vitro antiplasmodial activities of peptide antibiotics of fungal origin. The three peptide antibiotics used in this study were efrapeptins, zervamicins, and antiamoebin. The high-performance liquid chromatography-purified peptides were characterized by nuclear magnetic resonance and mass spectral analysis. All three fungal peptides kill P. falciparum in culture with 50% inhibitory concentrations in the micromolar range. A possible mode of action of these peptide antibiotics on P. falciparum is presented. PMID:11120957
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NASA Astrophysics Data System (ADS)
Khatri, Kshitij; Pu, Yi; Klein, Joshua A.; Wei, Juan; Costello, Catherine E.; Lin, Cheng; Zaia, Joseph
2018-04-01
Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be performed at almost the same scale as proteomics experiments. While collisionally activated dissociation (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of multiply glycosylated peptides. With improvements in instrumentation, electron-activated dissociation (ExD) modes are becoming increasingly prevalent for proteomics experiments and for the analysis of fragile modifications such as glycosylation. While these methods have been applied for glycopeptide analysis in isolated studies, an organized effort to compare their efficiencies, particularly for analysis of multiply glycosylated peptides (termed here middle-down glycoproteomics), has not been made. We therefore compared the performance of different ExD modes for middle-down glycopeptide analyses. We identified key features among the different dissociation modes and show that increased electron energy and supplemental activation provide the most useful data for middle-down glycopeptide analysis. [Figure not available: see fulltext.
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Kawa, Diane E; Stephens, Richard S
2002-05-15
The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.
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Panni, Simona; Montecchi-Palazzi, Luisa; Kiemer, Lars; Cabibbo, Andrea; Paoluzi, Serena; Santonico, Elena; Landgraf, Christiane; Volkmer-Engert, Rudolf; Bachi, Angela; Castagnoli, Luisa; Cesareni, Gianni
2011-01-01
Large-scale interaction studies contribute the largest fraction of protein interactions information in databases. However, co-purification of non-specific or indirect ligands, often results in data sets that are affected by a considerable number of false positives. For the fraction of interactions mediated by short linear peptides, we present here a combined experimental and computational strategy for ranking the reliability of the inferred partners. We apply this strategy to the family of 14-3-3 domains. We have first characterized the recognition specificity of this domain family, largely confirming the results of previous analyses, while revealing new features of the preferred sequence context of 14-3-3 phospho-peptide partners. Notably, a proline next to the carboxy side of the phospho-amino acid functions as a potent inhibitor of 14-3-3 binding. The position-specific information about residue preference was encoded in a scoring matrix and two regular expressions. The integration of these three features in a single predictive model outperforms publicly available prediction tools. Next we have combined, by a naïve Bayesian approach, these "peptide features" with "protein features", such as protein co-expression and co-localization. Our approach provides an orthogonal reliability assessment and maps with high confidence the 14-3-3 peptide target on the partner proteins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue
2013-01-01
We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization. PMID:23997661
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Bremer, A A; Leeman, S E; Boyd, N D
2000-12-01
Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.
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Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.
Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper
2016-10-10
Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations. Copyright © 2016 Elsevier B.V. All rights reserved.
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1985-01-01
A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary- shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co- linearly with the peptide chain. PMID:2413043
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Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.
Andreatta, Massimo; Lund, Ole; Nielsen, Morten
2013-01-01
Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides. The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities in peptide data by performing two essential tasks simultaneously: alignment and clustering of peptide data. We apply the method to de-convolute binding motifs in a panel of peptide datasets with different degrees of complexity spanning from the simplest case of pre-aligned fixed-length peptides to cases of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule. The Gibbs clustering method is available online as a web server at http://www.cbs.dtu.dk/services/GibbsCluster.
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Use of synthetic peptide libraries for the H-2Kd binding motif identification.
Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y
1995-01-01
To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.
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Yano, Akira; Ito, Kaori; Miwa, Yoshikatsu; Kanazawa, Yoshito; Chiba, Akiko; Iigo, Yutaka; Kashimoto, Yoshinori; Kanda, Akira; Murata, Shinji; Makino, Mitsuhiro
2015-01-01
The reduction of brain amyloid beta (Aβ) peptides by anti-Aβ antibodies is one of the possible therapies for Alzheimer's disease. We previously reported that the Aβ peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT) induced anti-Aβ antibodies, and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptide. Cynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination. Vaccination with a similar regimen was also performed on guinea pigs. The peptide vaccine induced anti-Aβ antibodies in cynomolgus monkeys and guinea pigs without chemical adjuvants, and excessive immune responses were not observed. Those antibodies could preferentially recognize Aβ 40, and Aβ 42 compared to Aβ fibrils. The levels of serum anti-Aβ antibodies and plasma Aβ peptides increased in both animals and decreased the brain Aβ 40 level of guinea pigs. The peptide vaccine could induce a similar binding profile of anti-Aβ antibodies in cynomolgus monkeys and guinea pigs. The peptide vaccination could be expected to reduce the brain Aβ peptides and their toxic effects via clearance of Aβ peptides by generated antibodies.
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An FPGA Implementation to Detect Selective Cationic Antibacterial Peptides
Polanco González, Carlos; Nuño Maganda, Marco Aurelio; Arias-Estrada, Miguel; del Rio, Gabriel
2011-01-01
Exhaustive prediction of physicochemical properties of peptide sequences is used in different areas of biological research. One example is the identification of selective cationic antibacterial peptides (SCAPs), which may be used in the treatment of different diseases. Due to the discrete nature of peptide sequences, the physicochemical properties calculation is considered a high-performance computing problem. A competitive solution for this class of problems is to embed algorithms into dedicated hardware. In the present work we present the adaptation, design and implementation of an algorithm for SCAPs prediction into a Field Programmable Gate Array (FPGA) platform. Four physicochemical properties codes useful in the identification of peptide sequences with potential selective antibacterial activity were implemented into an FPGA board. The speed-up gained in a single-copy implementation was up to 108 times compared with a single Intel processor cycle for cycle. The inherent scalability of our design allows for replication of this code into multiple FPGA cards and consequently improvements in speed are possible. Our results show the first embedded SCAPs prediction solution described and constitutes the grounds to efficiently perform the exhaustive analysis of the sequence-physicochemical properties relationship of peptides. PMID:21738652
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Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides.
Yu, Hua; Wang, Mao-jun; Xuan, Nan-xia; Shang, Zhi-cai; Wu, Jun
2015-10-01
To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues.
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[The antihypertensive effect of fermented milks].
Domínguez González, Karina N; Cruz Guerrero, Alma E; Márquez, Humberto González; Gómez Ruiz, Lorena C; García-Garibay, Mariano; Rodríguez Serrano, Gabriela M
2014-01-01
There is a great variety of fermented milks containing lactic acid bacteria that present health-promoting properties. Milk proteins are hydrolyzed by the proteolytic system of these microorganisms producing peptides which may also perform other functions in vivo. These peptides are encrypted within the primary structure of proteins and can be released through food processing, either by milk fermentation or enzymatic hydrolysis during gastrointestinal transit. They perform different activities, since they act in the cardiovascular, digestive, endocrine, immune and nervous systems. Bioactive peptides that have an antihypertensive, antithrombotic, antioxidant and hypocholesterolemic effect on the cardiovascular system can reduce the risk factors for chronic disease manifestation and help improve human health. Most studied bioactive peptides are those which exert an antihypertensive effect by inhibiting the angiotensin-converting enzyme (ACE). Recently, the study of these peptides has focused on the implementation of tests to prove that they have an effect on health. This paper focuses on the production of ACEinhibitory antihypertensive peptides from fermented milks, its history, production and in vivo tests on rats and humans, on which its hypotensive effect has been shown. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.
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Lamoliatte, Frederic; Bonneil, Eric; Durette, Chantal; Caron-Lizotte, Olivier; Wildemann, Dirk; Zerweck, Johannes; Wenshuk, Holger; Thibault, Pierre
2013-01-01
Protein modification by small ubiquitin-like modifier (SUMO) modulates the activities of numerous proteins involved in different cellular functions such as gene transcription, cell cycle, and DNA repair. Comprehensive identification of SUMOylated sites is a prerequisite to determine how SUMOylation regulates protein function. However, mapping SUMOylated Lys residues by mass spectrometry (MS) is challenging because of the dynamic nature of this modification, the existence of three functionally distinct human SUMO paralogs, and the large SUMO chain remnant that remains attached to tryptic peptides. To overcome these problems, we created HEK293 cell lines that stably express functional SUMO paralogs with an N-terminal His6-tag and an Arg residue near the C terminus that leave a short five amino acid SUMO remnant upon tryptic digestion. We determined the fragmentation patterns of our short SUMO remnant peptides by collisional activation and electron transfer dissociation using synthetic peptide libraries. Activation using higher energy collisional dissociation on the LTQ-Orbitrap Elite identified SUMO paralog-specific fragment ions and neutral losses of the SUMO remnant with high mass accuracy (< 5 ppm). We exploited these features to detect SUMO modified tryptic peptides in complex cell extracts by correlating mass measurements of precursor and fragment ions using a data independent acquisition method. We also generated bioinformatics tools to retrieve MS/MS spectra containing characteristic fragment ions to the identification of SUMOylated peptide by conventional Mascot database searches. In HEK293 cell extracts, this MS approach uncovered low abundance SUMOylated peptides and 37 SUMO3-modified Lys residues in target proteins, most of which were previously unknown. Interestingly, we identified mixed SUMO-ubiquitin chains with ubiquitylated SUMO proteins (K20 and K32) and SUMOylated ubiquitin (K63), suggesting a complex crosstalk between these two modifications. PMID:23750026
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Lazaridis, Themis; Leveritt, John M; PeBenito, Leo
2014-09-01
The energetic cost of burying charged groups in the hydrophobic core of lipid bilayers has been controversial, with simulations giving higher estimates than certain experiments. Implicit membrane approaches are usually deemed too simplistic for this problem. Here we challenge this view. The free energy of transfer of amino acid side chains from water to the membrane center predicted by IMM1 is reasonably close to all-atom free energy calculations. The shape of the free energy profile, however, for the charged side chains needs to be modified to reflect the all-atom simulation findings (IMM1-LF). Membrane thinning is treated by combining simulations at different membrane widths with an estimate of membrane deformation free energy from elasticity theory. This approach is first tested on the voltage sensor and the isolated S4 helix of potassium channels. The voltage sensor is stably inserted in a transmembrane orientation for both the original and the modified model. The transmembrane orientation of the isolated S4 helix is unstable in the original model, but a stable local minimum in IMM1-LF, slightly higher in energy than the interfacial orientation. Peptide translocation is addressed by mapping the effective energy of the peptide as a function of vertical position and tilt angle, which allows identification of minimum energy pathways and transition states. The barriers computed for the S4 helix and other experimentally studied peptides are low enough for an observable rate. Thus, computational results and experimental studies on the membrane burial of peptide charged groups appear to be consistent. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.
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C-type natriuretic peptide and atrial natriuretic peptide receptors of rat brain.
Brown, J; Zuo, Z
1993-03-01
Natriuretic peptide receptors in rat brain were mapped by in vitro autoradiography using 125I-labeled [Tyr0]CNP-(1-22) to bind atrial natriuretic peptide receptor (ANPR)-B and ANPR-C receptors selectively, and 125I-labeled alpha-ANP to select ANPR-A and ANPR-C receptors. Des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4- 23)-amide (C-ANP) was used for its selectivity for ANPR-C over ANPR-A. Specific binding of 125I-[Tyr0]CNP-(1-22) with a dissociation constant (Kd) approximately 1 nM occurred in olfactory bulb, cerebral cortex, lateral septal nucleus, choroid plexus, and arachnoid mater. This binding was abolished by C-type natriuretic peptide [CNP-(1-22)], alpha-ANP and C-ANP, and conformed to ANPR-C. 125I-alpha-ANP bound to all structures that bound 125I-[Tyr0]CNP-(1-22). This binding was also inhibited by both CNP-(1-22) and C-ANP, confirming the presence of ANPR-C-like binding sites. However, ANPR-C-like binding sites were heterogenous because only some had high affinities for 125I-[Tyr0]CNP-(1-22) and CNP-(1-22). 125I-alpha-ANP also bound sites without affinities for C-ANP or CNP-(1-22). These sites were consistent with ANPR-A. They occurred mainly on the olfactory bulb, the choroid plexus, and the subfornical organ. Guanosine 3',5'-cyclic monophosphate production was strongly stimulated by alpha-ANP but not by CNP-(1-22) in olfactory bulb. Neither ligand stimulated it in cortical tissue. Thus the natriuretic peptide binding sites of rat brain conformed to ANPR-A and to heterogenous ANPR-C-like sites. No ANPR-B were detected.
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Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.
Prickett, S R; Voskamp, A L; Phan, T; Dacumos-Hill, A; Mannering, S I; Rolland, J M; O'Hehir, R E
2013-06-01
Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (≤ 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays. Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations. © 2013 John Wiley & Sons Ltd.
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Zhang, Xi
2016-12-01
Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Mesri, E A; Levitus, G; Hontebeyrie-Joskowicz, M; Dighiero, G; Van Regenmortel, M H; Levin, M J
1990-01-01
A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease. PMID:1696282
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Nielsen, Morten; Lundegaard, Claus; Lund, Ole
2007-01-01
Background Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion The SMM-align method was shown to outperform other state of the art MHC class II prediction methods. The method predicts quantitative peptide:MHC binding affinity values, making it ideally suited for rational epitope discovery. The method has been trained and evaluated on the, to our knowledge, largest benchmark data set publicly available and covers the nine HLA-DR supertypes suggested as well as three mouse H2-IA allele. Both the peptide benchmark data set, and SMM-align prediction method (NetMHCII) are made publicly available. PMID:17608956
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Nielsen, Morten; Lundegaard, Claus; Lund, Ole
2007-07-04
Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. The SMM-align method was shown to outperform other state of the art MHC class II prediction methods. The method predicts quantitative peptide:MHC binding affinity values, making it ideally suited for rational epitope discovery. The method has been trained and evaluated on the, to our knowledge, largest benchmark data set publicly available and covers the nine HLA-DR supertypes suggested as well as three mouse H2-IA allele. Both the peptide benchmark data set, and SMM-align prediction method (NetMHCII) are made publicly available.
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PEPlife: A Repository of the Half-life of Peptides
NASA Astrophysics Data System (ADS)
Mathur, Deepika; Prakash, Satya; Anand, Priya; Kaur, Harpreet; Agrawal, Piyush; Mehta, Ayesha; Kumar, Rajesh; Singh, Sandeep; Raghava, Gajendra P. S.
2016-11-01
Short half-life is one of the key challenges in the field of therapeutic peptides. Various studies have reported enhancement in the stability of peptides using methods like chemical modifications, D-amino acid substitution, cyclization, replacement of labile aminos acids, etc. In order to study this scattered data, there is a pressing need for a repository dedicated to the half-life of peptides. To fill this lacuna, we have developed PEPlife (http://crdd.osdd.net/raghava/peplife), a manually curated resource of experimentally determined half-life of peptides. PEPlife contains 2229 entries covering 1193 unique peptides. Each entry provides detailed information of the peptide, like its name, sequence, half-life, modifications, the experimental assay for determining half-life, biological nature and activity of the peptide. We also maintain SMILES and structures of peptides. We have incorporated web-based modules to offer user-friendly data searching and browsing in the database. PEPlife integrates numerous tools to perform various types of analysis such as BLAST, Smith-Waterman algorithm, GGSEARCH, Jalview and MUSTANG. PEPlife would augment the understanding of different factors that affect the half-life of peptides like modifications, sequence, length, route of delivery of the peptide, etc. We anticipate that PEPlife will be useful for the researchers working in the area of peptide-based therapeutics.
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Overexpression of peptide deformylase in breast, colon, and lung cancers.
Randhawa, Harsharan; Chikara, Shireen; Gehring, Drew; Yildirim, Tuba; Menon, Jyotsana; Reindl, Katie M
2013-07-01
Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.
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Overexpression of peptide deformylase in breast, colon, and lung cancers
2013-01-01
Background Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. PMID:23815882
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Mikkonen, Saara; Jacksén, Johan; Roeraade, Johan; Thormann, Wolfgang; Emmer, Åsa
2016-10-18
A novel method for preconcentration and purification of the Alzheimer's disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Knight, K.L.; Hess, R.M.; McEntee, K.
1988-06-01
The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in eachmore » of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.« less
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Toward structure prediction of cyclic peptides.
Yu, Hongtao; Lin, Yu-Shan
2015-02-14
Cyclic peptides are a promising class of molecules that can be used to target specific protein-protein interactions. A computational method to accurately predict their structures would substantially advance the development of cyclic peptides as modulators of protein-protein interactions. Here, we develop a computational method that integrates bias-exchange metadynamics simulations, a Boltzmann reweighting scheme, dihedral principal component analysis and a modified density peak-based cluster analysis to provide a converged structural description for cyclic peptides. Using this method, we evaluate the performance of a number of popular protein force fields on a model cyclic peptide. All the tested force fields seem to over-stabilize the α-helix and PPII/β regions in the Ramachandran plot, commonly populated by linear peptides and proteins. Our findings suggest that re-parameterization of a force field that well describes the full Ramachandran plot is necessary to accurately model cyclic peptides.
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Ramezani, Fatemeh; Habibi, Mostafa; Rafii-Tabar, Hashem; Amanlou, Massoud
2015-01-29
Gold nanoparticles now command a great deal of attention for medical applications. Despite the importance of nano-bio interfaces, interaction between peptides and proteins with gold surfaces is not still fully understood, especially in a molecular level. In the present study computational simulation of adsorption of 20 amino acids, in three forms of mono-amino acid, homo di-peptide and homo tri-peptide, on the gold nanoparticles was performed by Gromacs using OPLSAA force field. The flexibility, stability, and size effect of the peptides on the gold nanoparticles were studied as well as the molecular structure of them. According to our results, adsorbed homo tri-peptides on the gold surface had more flexibility, more gyration, and the farthest distance from the GNP in comparison with homo di-peptides and mono-amino acids. Our findings provide new insights into the precise control of interactions between amino acids anchored on the GNPs.
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Method for enhanced accuracy in predicting peptides using liquid separations or chromatography
Kangas, Lars J.; Auberry, Kenneth J.; Anderson, Gordon A.; Smith, Richard D.
2006-11-14
A method for predicting the elution time of a peptide in chromatographic and electrophoretic separations by first providing a data set of known elution times of known peptides, then creating a plurality of vectors, each vector having a plurality of dimensions, and each dimension representing the elution time of amino acids present in each of these known peptides from the data set. The elution time of any protein is then be predicted by first creating a vector by assigning dimensional values for the elution time of amino acids of at least one hypothetical peptide and then calculating a predicted elution time for the vector by performing a multivariate regression of the dimensional values of the hypothetical peptide using the dimensional values of the known peptides. Preferably, the multivariate regression is accomplished by the use of an artificial neural network and the elution times are first normalized using a transfer function.
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sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides
DOE Office of Scientific and Technical Information (OSTI.GOV)
Luo, Heng; Ye, Hao; Ng, Hui Wen
Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less
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Aldrich, Jane V; Kulkarni, Santosh S; Senadheera, Sanjeewa N; Ross, Nicolette C; Reilley, Kate J; Eans, Shainnel O; Ganno, Michelle L; Murray, Thomas F; McLaughlin, Jay P
2011-09-05
An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides
Luo, Heng; Ye, Hao; Ng, Hui Wen; Sakkiah, Sugunadevi; Mendrick, Donna L.; Hong, Huixiao
2016-01-01
Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system. PMID:27558848
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sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides
Luo, Heng; Ye, Hao; Ng, Hui Wen; ...
2016-08-25
Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less
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Koutsopoulos, Sotirios
2016-04-01
Until the mid-1980s, mainly biologists were conducting peptide research. This changed with discoveries that opened new paths of research involving the use of peptides in bioengineering, biotechnology, biomedicine, nanotechnology, and bioelectronics. Peptide engineering and rational design of novel peptide sequences with unique and tailor-made properties further expanded the field. The discovery of short self-assembling peptides, which upon association form well-defined supramolecular architectures, created new and exciting areas of research. Depending on the amino acid sequence, the pH, and the type of the electrolyte in the medium, peptide self-assembly leads to the formation of nanofibers, which are further organized to form a hydrogel. In this review, the application of ionic complementary peptides which self-assemble to form nanofiber hydrogels for tissue engineering and regenerative medicine will be discussed through a selective presentation of the most important work performed during the last 25 years. © 2016 Wiley Periodicals, Inc.
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Huang, Junfeng; Qin, Hongqiang; Sun, Zhen; Huang, Guang; Mao, Jiawei; Cheng, Kai; Zhang, Zhang; Wan, Hao; Yao, Yating; Dong, Jing; Zhu, Jun; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa
2015-01-01
Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome. PMID:25959593
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Liu, Lianliang; Cao, Jinxuan; Chen, Jiong; Zhang, Xin; Wu, Zufang; Xiang, Huan
2016-09-19
This study was aimed to evaluate effects of peptides from Phascolosoma esculenta and its ferrous-chelating peptides on spatial learning and memory in mice by Morris water maze test. 100mg/kg peptide on spatial learning and memory function about quadrant time and passing times through the platform better than 50 and 150mg/kg group during exploration period (P<0.05), without body weight between the weight and visual ability. 100mg/kg ferrous-chelating peptide group performed better ability of spatial learning and memory than 100mg/kg peptide group (P<0.05). qRT-PCR results showed that 50 and 100mg/kg administration peptide and 100mg/kg ferrous-chelating peptide can significantly improve mRNA expression of NR2A, NR2B and BDNF with oxidative stress status (GSH-Px, SOD, TAC and MDA), which explained mechanism for improving learning and memory ability in mice via anti-oxidative character. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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Characterization of particulate matter binding peptides screened from phage display.
Liang Alvin, Aw Wei; Tanaka, Masayoshi; Okochi, Mina
2017-05-01
Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Jiang, Ying; Gao, Ge; Fang, Gang; Gustafson, Eric L; Laverty, Maureen; Yin, Yanbin; Zhang, Yong; Luo, Jingchu; Greene, Jonathan R; Bayne, Marvin L; Hedrick, Joseph A; Murgolo, Nicholas J
2003-05-01
PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. PepPat is offered online at: http://peppat.cbi.pku.edu.cn.
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Fan, Fengjiao; Tu, Maolin; Liu, Meng; Shi, Pujie; Wang, Yun; Wu, Di; Du, Ming
2017-08-23
Lactoferrin is reported to be a potential food protein with osteogenic activity. However, the activity of lactoferrin peptides is questionable. In the present study, we isolated and characterized peptides from lactoferrin with stimulatory effect on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were purified using cation-exchange and gel-filtration chromatography. Effects of different hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide 10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry confirmed that two peptides components of P5-a corresponded to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI: 221706349). These results will provide some theoretical and practical data for the preparation and application of osteogenic peptides in functional food industry.
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Peptide nanotube-modified electrodes for enzyme-biosensor applications.
Yemini, Miri; Reches, Meital; Gazit, Ehud; Rishpon, Judith
2005-08-15
The fabrication and notably improved performance of composite electrodes based on modified self-assembled diphenylalanine peptide nanotubes is described. Peptide nanotubes were attached to gold electrodes, and we studied the resulting electrochemical behavior using cyclic voltammetry and chronoamperometry. The peptide nanotube-based electrodes demonstrated a direct and unmediated response to hydrogen peroxide and NADH at a potential of +0.4 V (vs SCE). This biosensor enables a sensitive determination of glucose by monitoring the hydrogen peroxide produced by an enzymatic reaction between the glucose oxidase attached to the peptide nanotubes and glucose. In addition, the marked electrocatalytic activity toward NADH enabled a sensitive detection of ethanol using ethanol dehydrogenase and NAD+. The peptide nanotube-based amperometric biosensor provides a potential new tool for sensitive biosensors and biomolecular diagnostics.
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Shen, Bingquan; Zhang, Wanjun; Shi, Zhaomei; Tian, Fang; Deng, Yulin; Sun, Changqing; Wang, Guangshun; Qin, Weijie; Qian, Xiaohong
2017-07-01
O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine. Copyright © 2017. Published by Elsevier B.V.
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Tools for monitoring system suitability in LC MS/MS centric proteomic experiments.
Bereman, Michael S
2015-03-01
With advances in liquid chromatography coupled to tandem mass spectrometry technologies combined with the continued goals of biomarker discovery, clinical applications of established biomarkers, and integrating large multiomic datasets (i.e. "big data"), there remains an urgent need for robust tools to assess instrument performance (i.e. system suitability) in proteomic workflows. To this end, several freely available tools have been introduced that monitor a number of peptide identification (ID) and/or peptide ID free metrics. Peptide ID metrics include numbers of proteins, peptides, or peptide spectral matches identified from a complex mixture. Peptide ID free metrics include retention time reproducibility, full width half maximum, ion injection times, and integrated peptide intensities. The main driving force in the development of these tools is to monitor both intra- and interexperiment performance variability and to identify sources of variation. The purpose of this review is to summarize and evaluate these tools based on versatility, automation, vendor neutrality, metrics monitored, and visualization capabilities. In addition, the implementation of a robust system suitability workflow is discussed in terms of metrics, type of standard, and frequency of evaluation along with the obstacles to overcome prior to incorporating a more proactive approach to overall quality control in liquid chromatography coupled to tandem mass spectrometry based proteomic workflows. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.
Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver
2017-07-01
The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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de Medeiros, Sebastiao Freitas; Angelo, Laura Camila Antunes; de Medeiros, Matheus Antonio Souto; Banhara, Camila Regis; Barbosa, Bruna Barcelo; Yamamoto, Marcia Marly Winck
2018-01-01
Background The aim of this study was to examine the role of C-peptide as a biological marker of cardiometabolic risk in polycystic ovary syndrome (PCOS). Methods This case-control study enrolled 385 PCOS patients and 240 normal cycling women. Anthropometric and clinical variables were taken at first visit. Fasting C-peptide, glucose, lipids, and hormone measurements were performed. Simple and multiple correlations between C-peptide and other variables associated with dysmetabolism and cardiovascular disease were examined. Results C-peptide was well correlated with several anthropometric, metabolic, and endocrine parameters. In PCOS patients, stepwise multiple regression including C-peptide as the criterion variable and other predictors of cardiovascular disease risk provided a significant model in which the fasting C-peptide/glucose ratio, glucose, body weight, and free estrogen index (FEI) were retained (adjusted R2 = 0.988, F = 7.161, P = 0.008). Conclusion C-peptide levels alone or combined with C-peptide/glucose ratio, glucose, body weight, and FEI provided a significant model to identify PCOS patients with higher risk of future cardiometabolic diseases. PMID:29416587
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Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics
Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.
2009-01-01
The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504
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Carboxyalkylated Hemoglobin as a Potential Blood Substitute
1989-09-20
chromatography to remove minor and glycosylated hemoglobin components. Carbox) methylation Reaction - Many of the procedures have been described in our early...hemoglobin by peptide mapping after treatment with radiolabeled methyl acetyl phosphate. These binding sites are Met-l(3) and Lys-81(f) for liganded...ABSTRACT (Continue on reverse if necesary andia entify by block number) Carbox,, methylated hemoglobin is more stable than oxy hemoglobin during some
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USDA-ARS?s Scientific Manuscript database
Following a blood meal, Rhodnius prolixus undergoes a rapid diuresis in order to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas’ disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresi...
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A Protein Standard That Emulates Homology for the Characterization of Protein Inference Algorithms.
The, Matthew; Edfors, Fredrik; Perez-Riverol, Yasset; Payne, Samuel H; Hoopmann, Michael R; Palmblad, Magnus; Forsström, Björn; Käll, Lukas
2018-05-04
A natural way to benchmark the performance of an analytical experimental setup is to use samples of known composition and see to what degree one can correctly infer the content of such a sample from the data. For shotgun proteomics, one of the inherent problems of interpreting data is that the measured analytes are peptides and not the actual proteins themselves. As some proteins share proteolytic peptides, there might be more than one possible causative set of proteins resulting in a given set of peptides and there is a need for mechanisms that infer proteins from lists of detected peptides. A weakness of commercially available samples of known content is that they consist of proteins that are deliberately selected for producing tryptic peptides that are unique to a single protein. Unfortunately, such samples do not expose any complications in protein inference. Hence, for a realistic benchmark of protein inference procedures, there is a need for samples of known content where the present proteins share peptides with known absent proteins. Here, we present such a standard, that is based on E. coli expressed human protein fragments. To illustrate the application of this standard, we benchmark a set of different protein inference procedures on the data. We observe that inference procedures excluding shared peptides provide more accurate estimates of errors compared to methods that include information from shared peptides, while still giving a reasonable performance in terms of the number of identified proteins. We also demonstrate that using a sample of known protein content without proteins with shared tryptic peptides can give a false sense of accuracy for many protein inference methods.
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Gevaert, Bert; D'Hondt, Matthias; Bracke, Nathalie; Yao, Han; Wynendaele, Evelien; Vissers, Johannes Petrus Cornelis; De Cecco, Martin; Claereboudt, Jan; De Spiegeleer, Bart
2015-09-01
Cerebrolysin, a parenteral peptide preparation produced by controlled digestion of porcine brain proteins, is an approved nootropic medicine in some countries. However, it is also easily and globally available on the Internet. Nevertheless, until now, its exact chemical composition was unknown. Using high performance liquid chromatography (HPLC) coupled to ion trap and ultra high performance liquid chromatography (UHPLC) coupled to quadrupole-ion mobility-time-of-flight mass spectrometry (Q-IM-TOF MS), combined with UniProt pig protein database search and PEAKS de novo sequencing, we identified 638 unique peptides in an Internet-obtained Cerebrolysin sample. The main components in this sample originate from tubulin alpha- and beta-chain, actin, and myelin basic protein. No fragments of known neurotrophic factors like glial cell-derived neurotrophic factor (GDNF), neurotrophin nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) were found, suggesting that the activities reported in the literature are likely the result of new, hitherto unknown cryptic peptides with nootropic properties. Copyright © 2015 John Wiley & Sons, Ltd.
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Hynek, Radovan; Kuckova, Stepanka; Hradilova, Janka; Kodicek, Milan
2004-01-01
Identification of materials in color layers of paintings is necessary for correct decisions concerning restoration procedures as well as proving the authenticity of the painting. The proteins are usually important components of the painting layers. In this paper it has been demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be used for fast and reliable identification of proteins in color layers even in old, highly aged matrices. The digestion can be easily performed directly on silica wafers which are routinely used for infrared analysis. The amount of material necessary for such an analysis is extremely small. Peptide mass mapping using digestion with trypsin followed by MALDI-TOFMS and identification of the protein was successfully used for determination of the binder from a painting of the 19th century. Copyright 2004 John Wiley & Sons, Ltd.
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Chitta, Karnakar R; Landero-Figueroa, Julio A; Kodali, Phanichand; Caruso, Joseph A; Merino, Edward J
2013-09-30
Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS). Copyright © 2013 Elsevier B.V. All rights reserved.
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Bayes-Genis, Antoni; Barallat, Jaume; Galán, Amparo; de Antonio, Marta; Domingo, Mar; Zamora, Elisabet; Gastelurrutia, Paloma; Vila, Joan; Peñafiel, Judith; Gálvez-Montón, Carolina; Lupón, Josep
2015-12-01
Neprilysin breaks down numerous vasoactive peptides. The soluble form of neprilysin, which was recently identified in heart failure, is associated with cardiovascular outcomes. Within a multibiomarker strategy, we directly compared soluble neprilysin and N-terminal pro-B-type natriuretic peptide as risk stratifiers in a real-life cohort of heart failure patients. Soluble neprilysin, N-terminal pro-B-type natriuretic peptide, ST2, and high-sensitivity troponin T levels were measured in 797 consecutive ambulatory heart failure patients followed up for 4.7 years. Comprehensive multivariable analyses and soluble neprilysin vs N-terminal pro-B-type natriuretic peptide head-to-head assessments of performance were performed. A primary composite endpoint included cardiovascular death or heart failure hospitalization. A secondary endpoint explored cardiovascular death alone. Median soluble neprilysin and N-terminal pro-B-type natriuretic peptide concentrations were 0.64ng/mL and 1187 ng/L, respectively. Both biomarkers significantly correlated with age (P<.001) and ST2 (P<.001), but only N-terminal pro-B-type natriuretic peptide significantly correlated with estimated glomerular filtration rate (P<.001), body mass index (P<.001), left ventricular ejection fraction (P=.02) and high-sensitivity troponin T (P<.001). In multivariable Cox regression analyses, soluble neprilysin remained independently associated with the composite endpoint (hazard ratio=1.14; 95% confidence interval, 1.02-1.27; P=.03) and cardiovascular death (hazard ratio=1.15; 95% confidence interval, 1.01-1.31; P=.04), but N-terminal pro-B-type natriuretic peptide did not. The head-to-head soluble neprilysin vs N-terminal pro-B-type natriuretic peptide comparison showed good calibration and similar discrimination and reclassification for both neurohormonal biomarkers, but only soluble neprilysin improved overall goodness-of-fit. When added to a multimarker strategy, soluble neprilysin remained an independent prognosticator, while N-terminal pro-B-type natriuretic peptide lost significance as a risk stratifier in ambulatory patients with heart failure. Both biomarkers performed similarly in head-to-head analyses. Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.
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Tempest: Accelerated MS/MS database search software for heterogeneous computing platforms
Adamo, Mark E.; Gerber, Scott A.
2017-01-01
MS/MS database search algorithms derive a set of candidate peptide sequences from in-silico digest of a protein sequence database, and compute theoretical fragmentation patterns to match these candidates against observed MS/MS spectra. The original Tempest publication described these operations mapped to a CPU-GPU model, in which the CPU generates peptide candidates that are asynchronously sent to a discrete GPU to be scored against experimental spectra in parallel (Milloy et al., 2012). The current version of Tempest expands this model, incorporating OpenCL to offer seamless parallelization across multicore CPUs, GPUs, integrated graphics chips, and general-purpose coprocessors. Three protocols describe how to configure and run a Tempest search, including discussion of how to leverage Tempest's unique feature set to produce optimal results. PMID:27603022
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Pansieri, Jonathan; Halim, Mohammad A.; Vendrely, Charlotte; Dumoulin, Mireille; Legrand, François; Sallanon, Marcelle Moulin; Chierici, Sabine; Denti, Simona; Dagany, Xavier; Dugourd, Philippe; Marquette, Christel
2018-01-01
Heterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: Aβ1–42 peptide, tau and α-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population. In the case of Aβ1–42 peptide and tau protein, several coexisting species could be distinguished and characterized. In the case of α-synuclein, we show how the polymorphism affects the mass and charge distributions. PMID:29732065
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NASA Astrophysics Data System (ADS)
Dreisewerd, Klaus; Kingston, Robert; Geraerts, Wijnand P. M.; Li, Ka Wan
1997-12-01
Matrix-assisted laser desorption mass spectrometry (MALDI-MS) was performed directly on a small piece of single penis nerve of the pond snail, Lymnaea stagnalis, and reveals the presence of complex peptide profiles, including many hitherto undescribed peptides. Two of the peptides have molecular weights corresponding exactly to the previously described Lymnaea small cardioactive peptides (SCP) A and B. We confirmed their identities by structural characterization of the two peptides directly from a single penis nerve by matrix-assisted laser desorption ionization high-energy collision tandem MS analysis. MALDI-MS of nervous tissues also demonstrates that a cluster of central neurons, which send their axons to the penis nerve, contain the two peptides. As the penis nerve is the nerve that innervates the penis complex, we propose that the peptides are involved in the modulation of male copulatory processes. A bioassay indeed showed that the peptides increase the contraction frequency of the vas deference in a dose-dependent manner. The results demonstrate the potential of direct MALDI-MS analysis of nervous tissue to complement or substitute conventional biochemical techniques for the identification and localization of neuropeptides.
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Method for predicting peptide detection in mass spectrometry
Kangas, Lars [West Richland, WA; Smith, Richard D [Richland, WA; Petritis, Konstantinos [Richland, WA
2010-07-13
A method of predicting whether a peptide present in a biological sample will be detected by analysis with a mass spectrometer. The method uses at least one mass spectrometer to perform repeated analysis of a sample containing peptides from proteins with known amino acids. The method then generates a data set of peptides identified as contained within the sample by the repeated analysis. The method then calculates the probability that a specific peptide in the data set was detected in the repeated analysis. The method then creates a plurality of vectors, where each vector has a plurality of dimensions, and each dimension represents a property of one or more of the amino acids present in each peptide and adjacent peptides in the data set. Using these vectors, the method then generates an algorithm from the plurality of vectors and the calculated probabilities that specific peptides in the data set were detected in the repeated analysis. The algorithm is thus capable of calculating the probability that a hypothetical peptide represented as a vector will be detected by a mass spectrometry based proteomic platform, given that the peptide is present in a sample introduced into a mass spectrometer.
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99m Tc-HYNIC-(Ser)3 -J18 peptide: A radiotracer for non-small-cell lung cancer targeting.
Shaghaghi, Zahra; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal
2018-02-14
Radiolabeled peptide could be a useful tool for the diagnosis of non-small-cell lung cancer (NSCLC). In this study, HYNIC-(Ser) 3 -J18 peptide was labeled with 99m Tc using EDDA/tricine as coligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular-specific binding and tumor targeting in A-549 cells and tumor-bearing mice, respectively. The high radiochemical purity was obtained and this radiolabeled peptide exhibited high stability in buffer and serum. The radiolabeled peptide showed high affinity for the A-549 cells with a dissociation constant value (K D ) of 4.4 ± 0.8 nm. The tumor-muscles ratios were 2.7 and 4.4 at 1 and 2 hr after injection of 99m Tc-(EDDA/tricine)-HYNIC-(Ser) 3 -J18 in tumor-bearing mice. The tumor uptake was decreased after preinjection with non-labeled peptide for this radiolabeled peptide in blocking experiment. The results of this study showed the 99m Tc-(EDDA/tricine)-(Ser) 3 -HYNIC-J18 peptide might be a promising radiolabeled peptide for NSCLC targeting. © 2018 John Wiley & Sons A/S.
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Development of MRM-based assays for the absolute quantitation of plasma proteins.
Kuzyk, Michael A; Parker, Carol E; Domanski, Dominik; Borchers, Christoph H
2013-01-01
Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.
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Characterization, Preparation, and Purification of Marine Bioactive Peptides
Wang, Xueqin; Yu, Huahua; Xing, Ronge
2017-01-01
Marine bioactive peptides, as a source of unique bioactive compounds, are the focus of current research. They exert various biological roles, some of the most crucial of which are antioxidant activity, antimicrobial activity, anticancer activity, antihypertensive activity, anti-inflammatory activity, and so forth, and specific characteristics of the bioactivities are described. This review also describes various manufacturing techniques for marine bioactive peptides using organic synthesis, microwave assisted extraction, chemical hydrolysis, and enzymes hydrolysis. Finally, purification of marine bioactive peptides is described, including gel or size exclusion chromatography, ion-exchange column chromatography, and reversed-phase high-performance liquid chromatography, which are aimed at finding a fast, simple, and effective method to obtain the target peptides. PMID:28761878
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Entropic stabilization of isolated beta-sheets.
Dugourd, Philippe; Antoine, Rodolphe; Breaux, Gary; Broyer, Michel; Jarrold, Martin F
2005-04-06
Temperature-dependent electric deflection measurements have been performed for a series of unsolvated alanine-based peptides (Ac-WA(n)-NH(2), where Ac = acetyl, W = tryptophan, A = alanine, and n = 3, 5, 10, 13, and 15). The measurements are interpreted using Monte Carlo simulations performed with a parallel tempering algorithm. Despite alanine's high helix propensity in solution, the results suggest that unsolvated Ac-WA(n)-NH(2) peptides with n > 10 adopt beta-sheet conformations at room temperature. Previous studies have shown that protonated alanine-based peptides adopt helical or globular conformations in the gas phase, depending on the location of the charge. Thus, the charge more than anything else controls the structure.
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Hmrbase: a database of hormones and their receptors
Rashid, Mamoon; Singla, Deepak; Sharma, Arun; Kumar, Manish; Raghava, Gajendra PS
2009-01-01
Background Hormones are signaling molecules that play vital roles in various life processes, like growth and differentiation, physiology, and reproduction. These molecules are mostly secreted by endocrine glands, and transported to target organs through the bloodstream. Deficient, or excessive, levels of hormones are associated with several diseases such as cancer, osteoporosis, diabetes etc. Thus, it is important to collect and compile information about hormones and their receptors. Description This manuscript describes a database called Hmrbase which has been developed for managing information about hormones and their receptors. It is a highly curated database for which information has been collected from the literature and the public databases. The current version of Hmrbase contains comprehensive information about ~2000 hormones, e.g., about their function, source organism, receptors, mature sequences, structures etc. Hmrbase also contains information about ~3000 hormone receptors, in terms of amino acid sequences, subcellular localizations, ligands, and post-translational modifications etc. One of the major features of this database is that it provides data about ~4100 hormone-receptor pairs. A number of online tools have been integrated into the database, to provide the facilities like keyword search, structure-based search, mapping of a given peptide(s) on the hormone/receptor sequence, sequence similarity search. This database also provides a number of external links to other resources/databases in order to help in the retrieving of further related information. Conclusion Owing to the high impact of endocrine research in the biomedical sciences, the Hmrbase could become a leading data portal for researchers. The salient features of Hmrbase are hormone-receptor pair-related information, mapping of peptide stretches on the protein sequences of hormones and receptors, Pfam domain annotations, categorical browsing options, online data submission, DrugPedia linkage etc. Hmrbase is available online for public from . PMID:19589147
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Bommana, Sankhya; Walker, Evelyn; Desclozeaux, Marion; Timms, Peter; Polkinghorne, Adam
2017-01-01
Chlamydia pecorum is a globally recognised livestock pathogen due to the significant clinical and economic impact it poses to livestock producers. Routine serological diagnosis is through a complement fixation test (CFT), which is often criticised for cross-reactivity, poor sensitivity and specificity. Although serology remains the preferred method in veterinary diagnostic laboratories, serological assays based on surface antigens of C. pecorum have not been established until now. In this study, we evaluated the use of two chlamydial recombinant protein antigens (PmpG and MOMP-G) by a direct IgG ELISA method for detection of ovine anti-chlamydial antibodies. Using the Pepscan method we then identified B cell epitopes across PmpG and MOMP-G proteins, in lambs with (a) naturally occurring asymptomatic C. pecorum infections (b) C. pecorum-associated polyarthritis and (c) recombinant PmpG and MOMP-G vaccine. Plasma IgG antibodies to PmpG in natural infection of lambs were detected earlier in infection than CFT and served as an acute phase marker. Antibodies to MOMP-G IgG were significantly heightened in lambs with C. pecorum-associated polyarthritis. PmpG and MOMP-G specific B-cell epitope mapping revealed epitope responses in immunised lambs cluster with some of the epitope responses in naturally infected lambs. B-cell epitope mapping further revealed that lambs with polyarthritis recognised several unique PmpG (50% frequency, peptide 8, 25, 40, 41 and 50) and MOMP (50% frequency, peptide 50) epitopes in comparison to asymptomatic infections. The findings of this study will have implications towards improved serodiagnosis of C. pecorum infections in livestock and inform the downstream development of alternative peptide-based antigens for future C. pecorum vaccine studies.
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Cai, Kewen; Itoh, Yoshiki; Khorana, H. Gobind
2001-01-01
Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345. PMID:11320237
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Ambati, Aditya; Valentini, Davide; Montomoli, Emanuele; Lapini, Guilia; Biuso, Fabrizio; Wenschuh, Holger; Magalhaes, Isabelle; Maeurer, Markus
2015-01-01
A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix® vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251–265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine. PMID:25639813
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McMurray, M M; Hansen, J S; Haley, B E; Takemoto, D J; Takemoto, L J
1985-01-01
Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2983663
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The cardiovascular effects of a chimeric opioid peptide based on morphiceptin and PFRTic-NH2.
Li, Meixing; Zhou, Lanxia; Ma, Guoning; Cao, Shuo; Dong, Shouliang
2013-01-01
MCRT (YPFPFRTic-NH(2)) is a chimeric opioid peptide based on morphiceptin and PFRTic-NH(2). In order to assess the cardiovascular effect of MCRT, it was administered by intravenous (i.v.) injection targeting at the peripheral nervous system and by intracerebroventricular (i.c.v.) injection targeting at the central nervous system. Naloxone and L-NAME were injected before MCRT to investigate possible interactions with MCRT. Results show that administration of MCRT by i.v. or i.c.v. injection could induce bradycardia and decrease in mean arterial pressure (MAP) at a greater degree than that with morphiceptin and PFRTic-NH(2). When MCRT and NPFF were coinjected, we observed a dose-dependent weakening of these cardiovascular effects by MCRT. Because naloxone completely abolished the cardiovascular effects of MCRT, we conclude that opioid receptors are involved in regulating the MAP of MCRT regardless of modes of injection. The effect of MCRT on heart rate is completely dependent on opioid receptors when MCRT was administered by i.c.v. instead of i.v. The central nitric oxide (NO) pathway is involved in regulating blood pressure by MCRT under both modes of injection, but the peripheral NO pathway had no effect on lowering blood pressure mediated by MCRT when it was administered by i.c.v. Based on the results from different modes of injection, the regulation of heart rate by MCRT mainly involves in the central NO pathway. Lastly, we observed that the cardiovascular effects of MCRT such as bradycardia and decrease of blood pressure, were stronger than that of its parent peptides. Opioid receptors and the NO pathway are involved in the cardiovascular regulation by MCRT, and their degree of involvement differs between intravenous and intracerebroventricular injection. Copyright © 2012 Elsevier Inc. All rights reserved.
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Yano, Akira; Ito, Kaori; Miwa, Yoshikatsu; Kanazawa, Yoshito; Chiba, Akiko; Iigo, Yutaka; Kashimoto, Yoshinori; Kanda, Akira; Murata, Shinji; Makino, Mitsuhiro
2015-01-01
The reduction of brain amyloid beta (Aβ) peptides by anti-Aβ antibodies is one of the possible therapies for Alzheimer's disease. We previously reported that the Aβ peptide vaccine including the T-cell epitope of diphtheria-tetanus combined toxoid (DT) induced anti-Aβ antibodies, and the prior immunization with conventional DT vaccine enhanced the immunogenicity of the peptide. Cynomolgus monkeys were given the peptide vaccine subcutaneously in combination with the prior DT vaccination. Vaccination with a similar regimen was also performed on guinea pigs. The peptide vaccine induced anti-Aβ antibodies in cynomolgus monkeys and guinea pigs without chemical adjuvants, and excessive immune responses were not observed. Those antibodies could preferentially recognize Aβ 40, and Aβ 42 compared to Aβ fibrils. The levels of serum anti-Aβ antibodies and plasma Aβ peptides increased in both animals and decreased the brain Aβ 40 level of guinea pigs. The peptide vaccine could induce a similar binding profile of anti-Aβ antibodies in cynomolgus monkeys and guinea pigs. The peptide vaccination could be expected to reduce the brain Aβ peptides and their toxic effects via clearance of Aβ peptides by generated antibodies. PMID:26539559
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Li, Haiou; Lu, Liyao; Chen, Rong; Quan, Lijun; Xia, Xiaoyan; Lü, Qiang
2014-01-01
Structural information related to protein-peptide complexes can be very useful for novel drug discovery and design. The computational docking of protein and peptide can supplement the structural information available on protein-peptide interactions explored by experimental ways. Protein-peptide docking of this paper can be described as three processes that occur in parallel: ab-initio peptide folding, peptide docking with its receptor, and refinement of some flexible areas of the receptor as the peptide is approaching. Several existing methods have been used to sample the degrees of freedom in the three processes, which are usually triggered in an organized sequential scheme. In this paper, we proposed a parallel approach that combines all the three processes during the docking of a folding peptide with a flexible receptor. This approach mimics the actual protein-peptide docking process in parallel way, and is expected to deliver better performance than sequential approaches. We used 22 unbound protein-peptide docking examples to evaluate our method. Our analysis of the results showed that the explicit refinement of the flexible areas of the receptor facilitated more accurate modeling of the interfaces of the complexes, while combining all of the moves in parallel helped the constructing of energy funnels for predictions.
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Chen, Hui; Shi, Pujie; Fan, Fengjiao; Tu, Maolin; Xu, Zhe; Xu, Xianbing; Du, Ming
2018-05-01
Digested peptides of bovine lactoferrin as the functional hydrolysates were identified by the Q-TOF tandem mass spectrometry (Q-TOF-MS) coupled with ultra performance liquid chromatograph (UPLC) and capillary electrophoresis (CE). The former (UPLC-Q-TOF-MS) identified 106 peptides while the latter (CE-Q-TOF-MS) characterized 102 peptides after comparison of peptides in terms of their molecular weight (MW), mass-to-charge ratio (m/z), and isoelectric point (pI). In addition, the hydrophilic value, net charge (q), and molecular radius (r) of the peptides were calculated, and a correlation analysis of the two methods was conducted between the retention time (RT) and r/q ratio of the peptides in order to elucidate the different separation principles of the unique peptides. It was shown that the peptides with larger hydrophilic value were beneficial to be separated by UPLC, while the peptides with larger r/q ratio were beneficial to be separated by CE. Combination of the above mentioned two complementary techniques have confidently improved the sequence coverage of lactoferrin and enhanced the identification of peptides, which makes it up to 65.8% in this study. Copyright © 2018. Published by Elsevier B.V.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mannige, Ranjan V.
The Ramachandran plot is important to structural biology as it describes a peptide backbone in the context of its dominant degrees of freedom—the backbone dihedral anglesφandψ(Ramachandran, Ramakrishnan & Sasisekharan, 1963). Since its introduction, the Ramachandran plot has been a crucial tool to characterize protein backbone features. However, the conformation or twist of a backbone as a function ofφandψhas not been completely described for bothcisandtransbackbones. Additionally, little intuitive understanding is available about a peptide’s conformation simply from knowing theφandψvalues of a peptide (e.g., is the regular peptide defined byφ = ψ = - 100° left-handed or right-handed?). This report provides a new metric for backbone handednessmore » (h) based on interpreting a peptide backbone as a helix with axial displacementdand angular displacementθ, both of which are derived from a peptide backbone’s internal coordinates, especially dihedral anglesφ,ψandω. In particular,hequals sin(θ)d/d|, with range [-1, 1] and negative (or positive) values indicating left(or right)-handedness. The metrichis used to characterize the handedness of every region of the Ramachandran plot for bothcis(ω = 0°) and trans (ω = 180°) backbones, which provides the first exhaustive survey of twist handedness in Ramachandran (φ,ψ) space. These maps fill in the ‘dead space’ within the Ramachandran plot, which are regions that are not commonly accessed by structured proteins, but which may be accessible to intrinsically disordered proteins, short peptide fragments, and protein mimics such as peptoids. Finally, building on the work of (Zacharias & Knapp, 2013), this report presents a new plot based ondandθthat serves as a universal and intuitive alternative to the Ramachandran plot. The universality arises from the fact that the co-inhabitants of such a plot include every possible peptide backbone includingcisandtransbackbones. The intuitiveness arises from the fact thatdandθprovide, at a glance, numerous aspects of the backbone including compactness, handedness, and planarity.« less
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Mannige, Ranjan V.
2017-05-16
The Ramachandran plot is important to structural biology as it describes a peptide backbone in the context of its dominant degrees of freedom—the backbone dihedral anglesφandψ(Ramachandran, Ramakrishnan & Sasisekharan, 1963). Since its introduction, the Ramachandran plot has been a crucial tool to characterize protein backbone features. However, the conformation or twist of a backbone as a function ofφandψhas not been completely described for bothcisandtransbackbones. Additionally, little intuitive understanding is available about a peptide’s conformation simply from knowing theφandψvalues of a peptide (e.g., is the regular peptide defined byφ = ψ = - 100° left-handed or right-handed?). This report provides a new metric for backbone handednessmore » (h) based on interpreting a peptide backbone as a helix with axial displacementdand angular displacementθ, both of which are derived from a peptide backbone’s internal coordinates, especially dihedral anglesφ,ψandω. In particular,hequals sin(θ)d/d|, with range [-1, 1] and negative (or positive) values indicating left(or right)-handedness. The metrichis used to characterize the handedness of every region of the Ramachandran plot for bothcis(ω = 0°) and trans (ω = 180°) backbones, which provides the first exhaustive survey of twist handedness in Ramachandran (φ,ψ) space. These maps fill in the ‘dead space’ within the Ramachandran plot, which are regions that are not commonly accessed by structured proteins, but which may be accessible to intrinsically disordered proteins, short peptide fragments, and protein mimics such as peptoids. Finally, building on the work of (Zacharias & Knapp, 2013), this report presents a new plot based ondandθthat serves as a universal and intuitive alternative to the Ramachandran plot. The universality arises from the fact that the co-inhabitants of such a plot include every possible peptide backbone includingcisandtransbackbones. The intuitiveness arises from the fact thatdandθprovide, at a glance, numerous aspects of the backbone including compactness, handedness, and planarity.« less
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Shirangi, Mehrnoosh; Sastre Toraño, Javier; Sellergren, Börje; Hennink, Wim E; Somsen, Govert W; van Nostrum, Cornelus F
2015-01-21
Free radical polymerization is often used to prepare protein and peptide-loaded hydrogels for the design of controlled release systems and molecular imprinting materials. Peroxodisulfates (ammonium peroxodisulfates (APS) or potassium peroxodisulfates (KPS)) with N,N,N,N-tetramethylethylenediamine (TEMED) are frequently used as initiator and catalyst. However, exposure to these free radical polymerization reagents may lead to modification of the protein and peptide. In this work, we show the modification of lysine residues by ammonium peroxodisulfate (APS)/TEMED of the immunostimulant thymopentin (TP5). Parallel studies on a decapeptide and a library of 15 dipeptides were performed to reveal the mechanism of modification. LC-MS of APS/TEMED-exposed TP5 revealed a major reaction product with an increased mass (+12 Da) with respect to TP5. LC-MS(2) and LC-MS(3) were performed to obtain structural information on the modified peptide and localize the actual modification site. Interpretation of the obtained data demonstrates the formation of a methylene bridge between the lysine and arginine residue in the presence of TEMED, while replacing TEMED with a sodium bisulfite catalyst did not show this modification. Studies with the other peptides showed that the TEMED radical can induce methyleneation on peptides when lysine is next to arginine, proline, cysteine, aspargine, glutamine, histidine, tyrosine, tryptophan, and aspartic acid residues. Stability of peptides and protein needs to be considered when using APS/TEMED in in situ polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization.
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NASA Astrophysics Data System (ADS)
Ng, I.-Son; Yu, You-Jin; Yi, Ying-Chen; Tan, Shih-I.; Huang, Bo-Chuan; Han, Yin-Lung
2017-12-01
The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel gold binding peptide. Finally, the heterologous over-expression of gold binding peptide by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.
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Nakano, Miki; Watanabe, Hirofumi; Rothstein, Stuart M; Tanaka, Shigenori
2010-05-27
Polyglutamine (polyQ) diseases are caused by an abnormal expansion of CAG repeats. While their detailed structure remains unclear, polyQ peptides assume beta-sheet structures when they aggregate. To investigate the conformational ensemble of short, monomeric polyQ peptides, which consist of 15 glutamine residues (Q(15)), we performed replica exchange molecular dynamics (REMD) simulations. We found that Q(15) can assume multiple configurations due to all of the residues affecting the formation of side-chain hydrogen bonds. Analysis of the free energy landscape reveals that Q(15) has a basin for random-coil structures and another for alpha-helix or beta-turn structures. To investigate properties of aggregated polyQ peptides, we performed multiple molecular dynamics (MMD) simulations for monomeric and oligomeric Q(15). MMD revealed that the formation of oligomers stabilizes the beta-turn structure by increasing the number of hydrogen bonds between the main chains.