[Techniques for rapid production of monoclonal antibodies for use with antibody technology].

PubMed

Kamada, Haruhiko

2012-01-01

A monoclonal antibody (Mab), due to its specific binding ability to a target protein, can potentially be one of the most useful tools for the functional analysis of proteins in recent proteomics-based research. However, the production of Mab is a very time-consuming and laborious process (i.e., preparation of recombinant antigens, immunization of animals, preparation of hybridomas), making it the rate-limiting step in using Mabs in high-throughput proteomics research, which heavily relies on comprehensive and rapid methods. Therefore, there is a great demand for new methods to efficiently generate Mabs against a group of proteins identified by proteome analysis. Here, we describe a useful method called "Antibody proteomic technique" for the rapid generations of Mabs to pharmaceutical target, which were identified by proteomic analyses of disease samples (ex. tumor tissue, etc.). We also introduce another method to find profitable targets on vasculature, which is called "Vascular proteomic technique". Our results suggest that this method for the rapid generation of Mabs to proteins may be very useful in proteomics-based research as well as in clinical applications.

One Sample, One Shot - Evaluation of sample preparation protocols for the mass spectrometric proteome analysis of human bile fluid without extensive fractionation.

PubMed

Megger, Dominik A; Padden, Juliet; Rosowski, Kristin; Uszkoreit, Julian; Bracht, Thilo; Eisenacher, Martin; Gerges, Christian; Neuhaus, Horst; Schumacher, Brigitte; Schlaak, Jörg F; Sitek, Barbara

2017-02-10

The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context. Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Coupling detergent lysis/clean-up methodology with intact protein fractionation for enhanced proteome characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Ritin; Dill, Brian; Chourey, Karuna

2012-01-01

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four different detergent clean-up methods (Trichloroacetic acid (TCA) precipitation, Chloroform/Methanol/Water (CMW) extraction, commercial detergent removal spin column method (DRS) and filter-aided sample preparation(FASP)) with respect to varying amounts of protein biomass in the samples, and provide efficiency benchmarks with respect to protein, peptide, and spectral identifications for each method. Our results show that for protein limited samples, FASP outperforms the other three clean-up methods, while at high protein amountmore » all the methods are comparable. This information was used in a dual strategy of comparing molecular weight based fractionated and unfractionated lysates from three increasingly complex samples (Escherichia coli, a five microbial isolate mixture, and a natural microbial community groundwater sample), which were all lysed with SDS and cleaned up using FASP. The two approaches complemented each other by enhancing the number of protein identifications by 8%-25% across the three samples and provided broad pathway coverage.« less

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis.

PubMed

Lo, Andy; Tang, Yanan; Chen, Lu; Li, Liang

2013-07-25

Isotope labeling liquid chromatography-mass spectrometry (LC-MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomics of exhaled breath: methodological nuances and pitfalls.

PubMed

Kurova, Viktoria S; Anaev, Eldar C; Kononikhin, Alexey S; Fedorchenko, Kristina Yu; Popov, Igor A; Kalupov, Timothey L; Bratanov, Dmitriy O; Nikolaev, Eugenie N; Varfolomeev, Sergey D

2009-01-01

The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations.

Global Cell Proteome Profiling, Phospho-signaling and Quantitative 
Proteomics for Identification of New Biomarkers in Acute Myeloid 
Leukemia Patients

PubMed Central

Aasebø, Elise; Forthun, Rakel B.; Berven, Frode; Selheim, Frode; Hernandez-Valladares, Maria

2016-01-01

The identification of protein biomarkers for acute myeloid leukemia (AML) that could find applications in AML diagnosis and prognosis, treatment and the selection for bone marrow transplant requires substantial comparative analyses of the proteomes from AML patients. In the past years, several studies have suggested some biomarkers for AML diagnosis or AML classification using methods for sample preparation with low proteome coverage and low resolution mass spectrometers. However, most of the studies did not follow up, confirm or validate their candidates with more patient samples. Current proteomics methods, new high resolution and fast mass spectrometers allow the identification and quantification of several thousands of proteins obtained from few tens of μg of AML cell lysate. Enrichment methods for posttranslational modifications (PTM), such as phosphorylation, can isolate several thousands of site-specific phosphorylated peptides from AML patient samples, which subsequently can be quantified with high confidence in new mass spectrometers. While recent reports aiming to propose proteomic or phosphoproteomic biomarkers on the studied AML patient samples have taken advantage of the technological progress, the access to large cohorts of AML patients to sample from and the availability of appropriate control samples still remain challenging. PMID:26306748

Using HPLC-Mass Spectrometry to Teach Proteomics Concepts with Problem-Based Techniques

ERIC Educational Resources Information Center

Short, Michael; Short, Anne; Vankempen, Rachel; Seymour, Michael; Burnatowska-Hledin, Maria

2010-01-01

Practical instruction of proteomics concepts was provided using high-performance liquid chromatography coupled with a mass selective detection system (HPLC-MS) for the analysis of simulated protein digests. The samples were prepared from selected dipeptides in order to facilitate the mass spectral identification. As part of the prelaboratory…

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

2014-07-01

Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Facile preparation of salivary extracellular vesicles for cancer proteomics

NASA Astrophysics Data System (ADS)

Sun, Yan; Xia, Zhijun; Shang, Zhi; Sun, Kaibo; Niu, Xiaomin; Qian, Liqiang; Fan, Liu-Yin; Cao, Cheng-Xi; Xiao, Hua

2016-04-01

Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

Proteomic Challenges: Sample Preparation Techniques for Microgram-Quantity Protein Analysis from Biological Samples

PubMed Central

Feist, Peter; Hummon, Amanda B.

2015-01-01

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed. PMID:25664860

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

PubMed

Stachowicz, Aneta; Siudut, Jakub; Suski, Maciej; Olszanecki, Rafał; Korbut, Ryszard; Undas, Anetta; Wiśniewski, Jacek R

2017-01-01

It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

A Method for Label-Free, Differential Top-Down Proteomics.

PubMed

Ntai, Ioanna; Toby, Timothy K; LeDuc, Richard D; Kelleher, Neil L

2016-01-01

Biomarker discovery in the translational research has heavily relied on labeled and label-free quantitative bottom-up proteomics. Here, we describe a new approach to biomarker studies that utilizes high-throughput top-down proteomics and is the first to offer whole protein characterization and relative quantitation within the same experiment. Using yeast as a model, we report procedures for a label-free approach to quantify the relative abundance of intact proteins ranging from 0 to 30 kDa in two different states. In this chapter, we describe the integrated methodology for the large-scale profiling and quantitation of the intact proteome by liquid chromatography-mass spectrometry (LC-MS) without the need for metabolic or chemical labeling. This recent advance for quantitative top-down proteomics is best implemented with a robust and highly controlled sample preparation workflow before data acquisition on a high-resolution mass spectrometer, and the application of a hierarchical linear statistical model to account for the multiple levels of variance contained in quantitative proteomic comparisons of samples for basic and clinical research.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

PubMed

Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

2018-05-01

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf.

PubMed

Daim, Leona Daniela Jeffery; Ooi, Tony Eng Keong; Yusof, Hirzun Mohd; Majid, Nazia Abdul; Karsani, Saiful Anuar Bin

2015-08-01

Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.

Universal Solid-phase Reversible Sample-Prep for Concurrent Proteome and N-glycome Characterization

PubMed Central

Zhou, Hui; Morley, Samantha; Kostel, Stephen; Freeman, Michael R.; Joshi, Vivek; Brewster, David; Lee, Richard S.

2017-01-01

SUMMARY We describe a novel Solid-phase Reversible Sample-Prep (SRS) platform, which enables rapid sample preparation for concurrent proteome and N-glycome characterization by mass spectrometry. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal-to-no affinity for peptides and other small molecules. By leveraging the inherent size difference between, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, etc.), extensive manipulation including enzymatic and chemical treatments on beads-bound proteins, and easy recovery of N-glycans and peptides. The efficacy of SRS was evaluated in a wide range of biological samples including single glycoprotein, whole cell lysate, murine tissues, and human urine. To further demonstrate the SRS platform, we coupled a quantitative strategy to SRS to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Our previous studies suggested that DIAPH3 silencing in DU145 prostate cancer cells induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this analysis we identified distinct proteomic and N-glycomic alterations between the two cells. Intriguingly, a metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly upregulated in DIAPH3-silenced cells, indicating underling connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting a cross-link between DIAPH3 and glycosyltransferase networks. Overall, SRS is an enabling universal sample preparation strategy that is not size limited and has the capability to efficiently prepare and clean peptides and N-glycans concurrently from nearly all sample types. Conceptually, SRS can be utilized for the analysis of other posttranslational modifications, and the unique surface chemistry can be further transformed for high-throughput automation. The technical simplicity, robustness, and modularity of SRS make it a highly promising technology with great potential in proteomic-based research. PMID:26791391

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

PubMed Central

Li, Siyang; Plouffe, Brian D.; Belov, Arseniy M.; Ray, Somak; Wang, Xianzhe; Murthy, Shashi K.; Karger, Barry L.; Ivanov, Alexander R.

2015-01-01

Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. PMID:25755294

Microfluidic-Mass Spectrometry Interfaces for Translational Proteomics.

PubMed

Pedde, R Daniel; Li, Huiyan; Borchers, Christoph H; Akbari, Mohsen

2017-10-01

Interfacing mass spectrometry (MS) with microfluidic chips (μchip-MS) holds considerable potential to transform a clinician's toolbox, providing translatable methods for the early detection, diagnosis, monitoring, and treatment of noncommunicable diseases by streamlining and integrating laborious sample preparation workflows on high-throughput, user-friendly platforms. Overcoming the limitations of competitive immunoassays - currently the gold standard in clinical proteomics - μchip-MS can provide unprecedented access to complex proteomic assays having high sensitivity and specificity, but without the labor, costs, and complexities associated with conventional MS sample processing. This review surveys recent μchip-MS systems for clinical applications and examines their emerging role in streamlining the development and translation of MS-based proteomic assays by alleviating many of the challenges that currently inhibit widespread clinical adoption. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion.

PubMed

Wu, Shuaibin; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-10-30

A formic acid (FA)-assisted sample preparation method was presented for protein identification via mass spectrometry (MS). Detailedly, an aqueous solution containing 2% FA and dithiothreitol was selected to perform protein denaturation, aspartic acid (D) sites cleavage and disulfide linkages reduction simultaneously at 108°C for 2h. Subsequently, FA wiped off via vacuum concentration. Finally, iodoacetamide (IAA) alkylation and trypsin digestion could be performed ordinally. A series of model proteins (BSA, β-lactoglobulin and apo-Transferrin) were treated respectively using such method, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The identified peptide number was increased by ∼ 80% in comparison with the conventional urea-assisted sample preparation method. Moreover, BSA identification was achieved efficiently down to femtomole (25 ± 0 sequence coverage and 16 ± 1 peptides) via such method. In contrast, there were not peptides identified confidently via the urea-assisted method before desalination via the C18 zip tip. The absence of urea in this sample preparation method was an advantage for the more favorable digestion and MALDI-TOF MS analysis. The performances of two methods for the real sample (rat liver proteome) were also compared, followed by a nanoflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry system analysis. As a result, 1335 ± 43 peptides were identified confidently (false discovery rate <1%) via FA-assisted method, corresponding to 295 ± 12 proteins (of top match=1 and requiring 2 unique peptides at least). In contrast, there were only 1107 ± 16 peptides (corresponding to 231 ± 10 proteins) obtained from the conventional urea-assisted method. It was serving as a more efficient protein sample preparation method for researching specific proteomes better, and providing assistance to develop other proteomics analysis methods, such as, peptide quantitative analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics

PubMed Central

2015-01-01

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin. Here, we describe a FASP method adapted to 96-well filter plates, named 96FASP. Soluble urine concentrates containing ∼10 μg of total protein were processed by 96FASP and LC-MS resulting in 700–900 protein identifications at a 1% false discovery rate (FDR). The experimental repeatability, as assessed by label-free quantification and Pearson correlation analysis for shared proteins among replicates, was high (R ≥ 0.97). Application to urinary pellet lysates which is of particular interest in the context of urinary tract infection analysis was also demonstrated. On average, 1700 proteins (±398) were identified in five experiments. In a pilot study using 96FASP for analysis of eight soluble urine samples, we demonstrated that protein profiles of technical replicates invariably clustered; the protein profiles for distinct urine donors were very different from each other. Robust, highly parallel methods to generate peptide mixtures from urine and other body fluids are critical to increase cost-effectiveness in clinical proteomics projects. This 96FASP method has potential to become a gold standard for high-throughput quantitative clinical proteomics. PMID:24797144

Preparation of the low molecular weight serum proteome for mass spectrometry analysis.

PubMed

Waybright, Timothy J; Chan, King C; Veenstra, Timothy D; Xiao, Zhen

2013-01-01

The discovery of viable biomarkers or indicators of disease states is complicated by the inherent complexity of the chosen biological specimen. Every sample, whether it is serum, plasma, urine, tissue, cells, or a host of others, contains thousands of large and small components, each interacting in multiple ways. The need to concentrate on a group of these components to narrow the focus on a potential biomarker candidate becomes, out of necessity, a priority, especially in the search for immune-related low molecular weight serum biomarkers. One such method in the field of proteomics is to divide the sample proteome into groups based on the size of the protein, analyze each group, and mine the data for statistically significant items. This chapter details a portion of this method, concentrating on a method for fractionating and analyzing the low molecular weight proteome of human serum.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

PubMed

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

MStern Blotting-High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates.

PubMed

Berger, Sebastian T; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno

2015-10-01

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates*

PubMed Central

Berger, Sebastian T.; Ahmed, Saima; Muntel, Jan; Cuevas Polo, Nerea; Bachur, Richard; Kentsis, Alex; Steen, Judith; Steen, Hanno

2015-01-01

We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. In summary, our new method maintains the strengths of FASP and simultaneously overcomes one of the major limitations of FASP without compromising protein identification and quantification. PMID:26223766

A Straightforward and Highly Efficient Precipitation/On-pellet Digestion Procedure Coupled to a Long Gradient Nano-LC Separation and Orbitrap Mass Spectrometry for Label-free Expression Profiling of the Swine Heart Mitochondrial Proteome

PubMed Central

Duan, Xiaotao; Young, Rebecca; Straubinger, Robert M.; Page, Brian J.; Cao, Jin; Wang, Hao; Yu, Haoying; Canty, John M.; Qu, Jun

2009-01-01

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes. PMID:19290621

Placental Proteomics: A Shortcut to Biological Insight

PubMed Central

Robinson, John M.; Vandré, Dale D.; Ackerman, William E.

2012-01-01

Proteomics analysis of biological samples has the potential to identify novel protein expression patterns and/or changes in protein expression patterns in different developmental or disease states. An important component of successful proteomics research, at least in its present form, is to reduce the complexity of the sample if it is derived from cells or tissues. One method to simplify complex tissues is to focus on a specific, highly purified sub-proteome. Using this approach we have developed methods to prepare highly enriched fractions of the apical plasma membrane of the syncytiotrophoblast. Through proteomics analysis of this fraction we have identified over five hundred proteins several of which were previously not known to reside in the syncytiotrophoblast. Herein, we focus on two of these, dysferlin and myoferlin. These proteins, largely known from studies of skeletal muscle, may not have been found in the human placenta were it not for discovery-based proteomics analysis. This new knowledge, acquired through a discovery-driven approach, can now be applied for the generation of hypothesis-based experimentation. Thus discovery-based and hypothesis-based research are complimentary approaches that when coupled together can hasten scientific discoveries. PMID:19070895

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two-dimensional gel electrophoresis.

PubMed

Rodrigues, Silas Pessini; Ventura, José Aires; Zingali, R B; Fernandes, P M B

2009-01-01

A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.

Absolute Quantification of Middle- to High-Abundant Plasma Proteins via Targeted Proteomics.

PubMed

Dittrich, Julia; Ceglarek, Uta

2017-01-01

The increasing number of peptide and protein biomarker candidates requires expeditious and reliable quantification strategies. The utilization of liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) for the absolute quantitation of plasma proteins and peptides facilitates the multiplexed verification of tens to hundreds of biomarkers from smallest sample quantities. Targeted proteomics assays derived from bottom-up proteomics principles rely on the identification and analysis of proteotypic peptides formed in an enzymatic digestion of the target protein. This protocol proposes a procedure for the establishment of a targeted absolute quantitation method for middle- to high-abundant plasma proteins waiving depletion or enrichment steps. Essential topics as proteotypic peptide identification and LC-MS/MS method development as well as sample preparation and calibration strategies are described in detail.

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

PubMed

Bergkvist, Jonas; Ekström, Simon; Wallman, Lars; Löfgren, Mikael; Marko-Varga, György; Nilsson, Johan; Laurell, Thomas

2002-04-01

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

Optimizing of MALDI-ToF-based low-molecular-weight serum proteome pattern analysis in detection of breast cancer patients; the effect of albumin removal on classification performance.

PubMed

Pietrowska, M; Marczak, L; Polanska, J; Nowicka, E; Behrent, K; Tarnawski, R; Stobiecki, M; Polanski, A; Widlak, P

2010-01-01

Mass spectrometry-based analysis of the serum proteome allows identifying multi-peptide patterns/signatures specific for blood of cancer patients, thus having high potential value for cancer diagnostics. However, because of problems with optimization and standardization of experimental and computational design, none of identified proteome patterns/signatures was approved for diagnostics in clinical practice as yet. Here we compared two methods of serum sample preparation for mass spectrometry-based proteome pattern analysis aimed to identify biomarkers that could be used in early detection of breast cancer patients. Blood samples were collected in a group of 92 patients diagnosed at early (I and II) stages of the disease before the start of therapy, and in a group of age-matched healthy controls (104 women). Serum specimens were purified and analyzed using MALDI-ToF spectrometry, either directly or after membrane filtration (50 kDa cut-off) to remove albumin and other large serum proteins. Mass spectra of the low-molecular-weight fraction (2-10 kDa) of the serum proteome were resolved using the Gaussian mixture decomposition, and identified spectral components were used to build classifiers that differentiated samples from breast cancer patients and healthy persons. Mass spectra of complete serum and membrane-filtered albumin-depleted samples have apparently different structure and peaks specific for both types of samples could be identified. The optimal classifier built for the complete serum specimens consisted of 8 spectral components, and had 81% specificity and 72% sensitivity, while that built for the membrane-filtered samples consisted of 4 components, and had 80% specificity and 81% sensitivity. We concluded that pre-processing of samples to remove albumin might be recommended before MALDI-ToF mass spectrometric analysis of the low-molecular-weight components of human serum Keywords: albumin removal; breast cancer; clinical proteomics; mass spectrometry; pattern analysis; serum proteome.

The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis

PubMed Central

Zou, Lili; Shen, Kaini; Zhong, Dingrong; Zhou, Daobin; Sun, Wei; Li, Jian

2015-01-01

Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples. PMID:25984759

PRESENTATION TYPE: Round Table Discussion (80 minutes) TITLE: Unlocking the ‘Omics Archive: Enabling Toxicogenomic/Proteomic Investigation from Archival Samples

EPA Science Inventory

Formalin fixation and paraffin embedding (FFPE) is a cross-industry gold standard for preparing nonclinical and clinical samples for histopathological assessment which preserves tissue architecture and enables storage of tissue in archival banks. These archival banks are an untap...

Sample handling for mass spectrometric proteomic investigations of human sera.

PubMed

West-Nielsen, Mikkel; Høgdall, Estrid V; Marchiori, Elena; Høgdall, Claus K; Schou, Christian; Heegaard, Niels H H

2005-08-15

Proteomic investigations of sera are potentially of value for diagnosis, prognosis, choice of therapy, and disease activity assessment by virtue of discovering new biomarkers and biomarker patterns. Much debate focuses on the biological relevance and the need for identification of such biomarkers while less effort has been invested in devising standard procedures for sample preparation and storage in relation to model building based on complex sets of mass spectrometric (MS) data. Thus, development of standardized methods for collection and storage of patient samples together with standards for transportation and handling of samples are needed. This requires knowledge about how sample processing affects MS-based proteome analyses and thereby how nonbiological biased classification errors are avoided. In this study, we characterize the effects of sample handling, including clotting conditions, storage temperature, storage time, and freeze/thaw cycles, on MS-based proteomics of human serum by using principal components analysis, support vector machine learning, and clustering methods based on genetic algorithms as class modeling and prediction methods. Using spiking to artificially create differentiable sample groups, this integrated approach yields data that--even when working with sample groups that differ more than may be expected in biological studies--clearly demonstrate the need for comparable sampling conditions for samples used for modeling and for the samples that are going into the test set group. Also, the study emphasizes the difference between class prediction and class comparison studies as well as the advantages and disadvantages of different modeling methods.

A review on recent developments in mass spectrometry instrumentation and quantitative tools advancing bacterial proteomics.

PubMed

Van Oudenhove, Laurence; Devreese, Bart

2013-06-01

Proteomics has evolved substantially since its early days, some 20 years ago. In this mini-review, we aim to provide an overview of general methodologies and more recent developments in mass spectrometric approaches used for relative and absolute quantitation of proteins. Enhancement of sensitivity of the mass spectrometers as well as improved sample preparation and protein fractionation methods are resulting in a more comprehensive analysis of proteomes. We also document some upcoming trends for quantitative proteomics such as the use of label-free quantification methods. Hopefully, microbiologists will continue to explore proteomics as a tool in their research to understand the adaptation of microorganisms to their ever changing environment. We encourage them to incorporate some of the described new developments in mass spectrometry to facilitate their analyses and improve the general knowledge of the fascinating world of microorganisms.

Proteomic platform for the identification of proteins in olive (Olea europaea) pulp.

PubMed

Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Piovesana, Susy; Samperi, Roberto; Stampachiacchiere, Serena; Laganà, Aldo

2013-10-24

The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues. Copyright © 2013. Published by Elsevier B.V.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

NASA Astrophysics Data System (ADS)

Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

2013-08-01

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Wheat proteomics: proteome modulation and abiotic stress acclimation

PubMed Central

Komatsu, Setsuko; Kamal, Abu H. M.; Hossain, Zahed

2014-01-01

Cellular mechanisms of stress sensing and signaling represent the initial plant responses to adverse conditions. The development of high-throughput “Omics” techniques has initiated a new era of the study of plant molecular strategies for adapting to environmental changes. However, the elucidation of stress adaptation mechanisms in plants requires the accurate isolation and characterization of stress-responsive proteins. Because the functional part of the genome, namely the proteins and their post-translational modifications, are critical for plant stress responses, proteomic studies provide comprehensive information about the fine-tuning of cellular pathways that primarily involved in stress mitigation. This review summarizes the major proteomic findings related to alterations in the wheat proteomic profile in response to abiotic stresses. Moreover, the strengths and weaknesses of different sample preparation techniques, including subcellular protein extraction protocols, are discussed in detail. The continued development of proteomic approaches in combination with rapidly evolving bioinformatics tools and interactive databases will facilitate understanding of the plant mechanisms underlying stress tolerance. PMID:25538718

A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.

PubMed

Bengtsson, Oskar; Arntzen, Magnus Ø; Mathiesen, Geir; Skaugen, Morten; Eijsink, Vincent G H

2016-01-10

Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates. Copyright © 2015 Elsevier B.V. All rights reserved.

Experimental Null Method to Guide the Development of Technical Procedures and to Control False-Positive Discovery in Quantitative Proteomics.

PubMed

Shen, Xiaomeng; Hu, Qiang; Li, Jun; Wang, Jianmin; Qu, Jun

2015-10-02

Comprehensive and accurate evaluation of data quality and false-positive biomarker discovery is critical to direct the method development/optimization for quantitative proteomics, which nonetheless remains challenging largely due to the high complexity and unique features of proteomic data. Here we describe an experimental null (EN) method to address this need. Because the method experimentally measures the null distribution (either technical or biological replicates) using the same proteomic samples, the same procedures and the same batch as the case-vs-contol experiment, it correctly reflects the collective effects of technical variability (e.g., variation/bias in sample preparation, LC-MS analysis, and data processing) and project-specific features (e.g., characteristics of the proteome and biological variation) on the performances of quantitative analysis. To show a proof of concept, we employed the EN method to assess the quantitative accuracy and precision and the ability to quantify subtle ratio changes between groups using different experimental and data-processing approaches and in various cellular and tissue proteomes. It was found that choices of quantitative features, sample size, experimental design, data-processing strategies, and quality of chromatographic separation can profoundly affect quantitative precision and accuracy of label-free quantification. The EN method was also demonstrated as a practical tool to determine the optimal experimental parameters and rational ratio cutoff for reliable protein quantification in specific proteomic experiments, for example, to identify the necessary number of technical/biological replicates per group that affords sufficient power for discovery. Furthermore, we assessed the ability of EN method to estimate levels of false-positives in the discovery of altered proteins, using two concocted sample sets mimicking proteomic profiling using technical and biological replicates, respectively, where the true-positives/negatives are known and span a wide concentration range. It was observed that the EN method correctly reflects the null distribution in a proteomic system and accurately measures false altered proteins discovery rate (FADR). In summary, the EN method provides a straightforward, practical, and accurate alternative to statistics-based approaches for the development and evaluation of proteomic experiments and can be universally adapted to various types of quantitative techniques.

NMR in the SPINE Structural Proteomics project.

PubMed

Ab, E; Atkinson, A R; Banci, L; Bertini, I; Ciofi-Baffoni, S; Brunner, K; Diercks, T; Dötsch, V; Engelke, F; Folkers, G E; Griesinger, C; Gronwald, W; Günther, U; Habeck, M; de Jong, R N; Kalbitzer, H R; Kieffer, B; Leeflang, B R; Loss, S; Luchinat, C; Marquardsen, T; Moskau, D; Neidig, K P; Nilges, M; Piccioli, M; Pierattelli, R; Rieping, W; Schippmann, T; Schwalbe, H; Travé, G; Trenner, J; Wöhnert, J; Zweckstetter, M; Kaptein, R

2006-10-01

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation.

PubMed

Cheng, Dongwan; Zheng, Li; Hou, Junjie; Wang, Jifeng; Xue, Peng; Yang, Fuquan; Xu, Tao

2015-01-01

The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

C-STrap Sample Preparation Method--In-Situ Cysteinyl Peptide Capture for Bottom-Up Proteomics Analysis in the STrap Format.

PubMed

Zougman, Alexandre; Banks, Rosamonde E

2015-01-01

Recently we introduced the concept of Suspension Trapping (STrap) for bottom-up proteomics sample processing that is based upon SDS-mediated protein extraction, swift detergent removal and rapid reactor-type protein digestion in a quartz depth filter trap. As the depth filter surface is made of silica, it is readily modifiable with various functional groups using the silane coupling chemistries. Thus, during the digest, peptides possessing specific features could be targeted for enrichment by the functionalized depth filter material while non-targeted peptides could be collected as an unbound distinct fraction after the digest. In the example presented here the quartz depth filter surface is functionalized with the pyridyldithiol group therefore enabling reversible in-situ capture of the cysteine-containing peptides generated during the STrap-based digest. The described C-STrap method retains all advantages of the original STrap methodology and provides robust foundation for the conception of the targeted in-situ peptide fractionation in the STrap format for bottom-up proteomics. The presented data support the method's use in qualitative and semi-quantitative proteomics experiments.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

PubMed Central

Rocker, Jana M; Tan, Marcus C; Thompson, Lee W; Contreras, Carlo M; DiPalma, Jack A; Pannell, Lewis K

2016-01-01

OBJECTIVES: There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. METHODS: WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. RESULTS: We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. CONCLUSIONS: WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer. PMID:27228405

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis

PubMed Central

Padula, Matthew P.; Berry, Iain J.; O′Rourke, Matthew B.; Raymond, Benjamin B.A.; Santos, Jerran; Djordjevic, Steven P.

2017-01-01

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer. PMID:28387712

A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.

PubMed

Padula, Matthew P; Berry, Iain J; O Rourke, Matthew B; Raymond, Benjamin B A; Santos, Jerran; Djordjevic, Steven P

2017-04-07

Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform's abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism's proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.

Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

PubMed Central

Zhang, Xi; Miller, Keith W.

2015-01-01

The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-β-d-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses—without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. PMID:25473089

Comparing Simplification Strategies for the Skeletal Muscle Proteome

PubMed Central

Geary, Bethany; Young, Iain S.; Cash, Phillip; Whitfield, Phillip D.; Doherty, Mary K.

2016-01-01

Skeletal muscle is a complex tissue that is dominated by the presence of a few abundant proteins. This wide dynamic range can mask the presence of lower abundance proteins, which can be a confounding factor in large-scale proteomic experiments. In this study, we have investigated a number of pre-fractionation methods, at both the protein and peptide level, for the characterization of the skeletal muscle proteome. The analyses revealed that the use of OFFGEL isoelectric focusing yielded the largest number of protein identifications (>750) compared to alternative gel-based and protein equalization strategies. Further, OFFGEL led to a substantial enrichment of a different sub-population of the proteome. Filter-aided sample preparation (FASP), coupled to peptide-level OFFGEL provided more confidence in the results due to a substantial increase in the number of peptides assigned to each protein. The findings presented here support the use of a multiplexed approach to proteome characterization of skeletal muscle, which has a recognized imbalance in the dynamic range of its protein complement. PMID:28248220

Proteomic Profiling of Skin Fibroblasts as a Model of Schizophrenia.

PubMed

Wang, Lan; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Since many aspects of schizophrenia are also manifested at the peripheral level in proliferating cell types, this chapter describes the analysis of skin fibroblasts biopsied from living patients. The method focuses on cell culture and sample preparation for characterization of the model. The resulting cell extracts can be analysed by any number of proteomic techniques for identification of biomarker candidates. This approach could help to elucidate the molecular mechanisms associated with the pathophysiology of schizophrenia and provide a useful model for a new target and drug discovery.

Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe.

PubMed

He, Sizhi; Wang, Qingsong; He, Jintang; Pu, Hai; Yang, Wei; Ji, Jianguo

2006-09-01

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue*

PubMed Central

MacDonald, Matthew L.; Ciccimaro, Eugene; Prakash, Amol; Banerjee, Anamika; Seeholzer, Steven H.; Blair, Ian A.; Hahn, Chang-Gyu

2012-01-01

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. PMID:22942359

Postmenopausal estrogen and progestin effects on the serum proteome

PubMed Central

2009-01-01

Background Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated. Methods Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool. Results In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates <0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways. Conclusions Serum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects. PMID:20034393

Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions

USDA-ARS?s Scientific Manuscript database

Initial screening for bacteriophages lytic for Clostridium perfringens was performed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Lytic phage preparations were initially characterized by transmission electron microscopy ...

Reference Proteome Extracts for Mass Spec Instrument Performance Validation and Method Development

PubMed Central

Rosenblatt, Mike; Urh, Marjeta; Saveliev, Sergei

2014-01-01

Biological samples of high complexity are required to test protein mass spec sample preparation procedures and validate mass spec instrument performance. Total cell protein extracts provide the needed sample complexity. However, to be compatible with mass spec applications, such extracts should meet a number of design requirements: compatibility with LC/MS (free of detergents, etc.)high protein integrity (minimal level of protein degradation and non-biological PTMs)compatibility with common sample preparation methods such as proteolysis, PTM enrichment and mass-tag labelingLot-to-lot reproducibility Here we describe total protein extracts from yeast and human cells that meet the above criteria. Two extract formats have been developed: Intact protein extracts with primary use for sample preparation method development and optimizationPre-digested extracts (peptides) with primary use for instrument validation and performance monitoring

Plant iTRAQ-based proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Handakumbura, Pubudu; Hixson, Kim K.; Purvine, Samuel O.

We present a simple one-­pot extraction protocol, which rapidly isolates hydrophyllic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlinedmore » here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z-­score values, which normalize the expression data into a format that can easily be cross-­compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation.« less

Simultaneous extraction of proteins and metabolites from cells in culture

PubMed Central

Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

2014-01-01

Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938

Biomarkers for pulmonary injury following deployment

DTIC Science & Technology

2013-10-01

construed as an official Department of the Army position, policy or decision unless so designated by other documentation. 2 REPORT DOCUMENTATION...cc x 3). BALF sample preparation for proteome analysis. Samples were thawed and desalted and concentrated with Amicon 3K MWCO spin filters (EMD...of the correlation heatmaps designate control and disease, respectively. Figure 2. PCA plot of BALF and urine datasets on left and right

A high-throughput semi-automated preparation for filtered synaptoneurosomes.

PubMed

Murphy, Kathryn M; Balsor, Justin; Beshara, Simon; Siu, Caitlin; Pinto, Joshua G A

2014-09-30

Synaptoneurosomes have become an important tool for studying synaptic proteins. The filtered synaptoneurosomes preparation originally developed by Hollingsworth et al. (1985) is widely used and is an easy method to prepare synaptoneurosomes. The hand processing steps in that preparation, however, are labor intensive and have become a bottleneck for current proteomic studies using synaptoneurosomes. For this reason, we developed new steps for tissue homogenization and filtration that transform the preparation of synaptoneurosomes to a high-throughput, semi-automated process. We implemented a standardized protocol with easy to follow steps for homogenizing multiple samples simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, LLC) and then filtering all of the samples in centrifugal filter units (EMD Millipore, Corp). The new steps dramatically reduce the time to prepare synaptoneurosomes from hours to minutes, increase sample recovery, and nearly double enrichment for synaptic proteins. These steps are also compatible with biosafety requirements for working with pathogen infected brain tissue. The new high-throughput semi-automated steps to prepare synaptoneurosomes are timely technical advances for studies of low abundance synaptic proteins in valuable tissue samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Proteomics of Skeletal Muscle: Focus on Insulin Resistance and Exercise Biology

PubMed Central

Deshmukh, Atul S.

2016-01-01

Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence, of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle proteomics are challenging. This review describes the technical limitations of skeletal muscle proteomics as well as emerging developments in proteomics workflow with respect to samples preparation, liquid chromatography (LC), MS and computational analysis. These technologies have not yet been fully exploited in the field of skeletal muscle proteomics. Future studies that involve state-of-the-art proteomics technology will broaden our understanding of exercise-induced adaptations as well as molecular pathogenesis of insulin resistance. This could lead to the identification of new therapeutic targets. PMID:28248217

Stable isotope dimethyl labelling for quantitative proteomics and beyond

PubMed Central

Hsu, Jue-Liang; Chen, Shu-Hui

2016-01-01

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

Proteomics Analysis of Skeletal Muscle from Leptin-Deficient ob/ob Mice Reveals Adaptive Remodeling of Metabolic Characteristics and Fiber Type Composition.

PubMed

Schönke, Milena; Björnholm, Marie; Chibalin, Alexander V; Zierath, Juleen R; Deshmukh, Atul S

2018-03-01

Skeletal muscle insulin resistance, an early metabolic defect in the pathogenesis of type 2 diabetes (T2D), may be a cause or consequence of altered protein expression profiles. Proteomics technology offers enormous promise to investigate molecular mechanisms underlying pathologies, however, the analysis of skeletal muscle is challenging. Using state-of-the-art multienzyme digestion and filter-aided sample preparation (MED-FASP) and a mass spectrometry (MS)-based workflow, we performed a global proteomics analysis of skeletal muscle from leptin-deficient, obese, insulin resistant (ob/ob) and lean mice in mere two fractions in a short time (8 h per sample). We identified more than 6000 proteins with 118 proteins differentially regulated in obesity. This included protein kinases, phosphatases, and secreted and fiber type associated proteins. Enzymes involved in lipid metabolism in skeletal muscle from ob/ob mice were increased, providing evidence against reduced fatty acid oxidation in lipid-induced insulin resistance. Mitochondrial and peroxisomal proteins, as well as components of pyruvate and lactate metabolism, were increased. Finally, the skeletal muscle proteome from ob/ob mice displayed a shift toward the "slow fiber type." This detailed characterization of an obese rodent model of T2D demonstrates an efficient workflow for skeletal muscle proteomics, which may easily be adapted to other complex tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics of industrial fungi: trends and insights for biotechnology.

PubMed

de Oliveira, José Miguel P Ferreira; de Graaff, Leo H

2011-01-01

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.

Proteomics of industrial fungi: trends and insights for biotechnology

PubMed Central

de Oliveira, José Miguel P. Ferreira

2010-01-01

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis. PMID:20922379

Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets.

PubMed

Zhu, Ying; Dou, Maowei; Piehowski, Paul D; Liang, Yiran; Wang, Fangjun; Chu, Rosalie K; Chrisler, Will; Smith, Jordan N; Schwarz, Kaitlynn C; Shen, Yufeng; Shukla, Anil K; Moore, Ronald J; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-06-24

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution due to the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 µm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-µm-thick rat brain cortex tissue sections with diameters of 50, 100, and 200 µm, respectively. We also analyzed 100-µm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ~1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.

PubMed

Prasad, Bhagwat; Unadkat, Jashvant D

2014-07-01

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.

Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.

2015-08-07

Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extractionmore » improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.« less

[Building Mass Spectrometry Spectral Libraries of Human Cancer Cell Lines].

PubMed

Faktor, J; Bouchal, P

Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.Key words: biomarker - mass spectrometry - proteomics - digital biobanking - SWATH - protein quantificationThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 7. 5. 2016Accepted: 9. 6. 2016.

Applications of reversible covalent chemistry in analytical sample preparation.

PubMed

Siegel, David

2012-12-07

Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes

PubMed Central

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-01-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)1 not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. PMID:27215607

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

PubMed

Virant-Klun, Irma; Leicht, Stefan; Hughes, Christopher; Krijgsveld, Jeroen

2016-08-01

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic features of delayed ocular symptoms caused by exposure to sulfur mustard: As studied by protein profiling of corneal epithelium.

PubMed

Pajoohesh, Maryam; Naderi, Mostafa; Naderi-Manesh, Hossein

2017-11-01

Exposure to mustard gas can lead to variations in the proteome of corneal epithelium cells and after a latency period produces delayed symptoms in the eyes of chemical victims. Hence, a comparative proteome analysis was conducted between the corneal epithelial cells of chemical victims from Iran-Iraq war (1980-1988) and healthy donors. To this end, corneal epithelium samples from victims and healthy individuals were collected, and the proteome of these samples were prepared for two-dimensional electrophoresis and the analysis of spots by statistical software. Selected spots were further analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Twenty four proteins were identified of which eighteen proteins showed downregulation while six proteins were upregulated in the victims in comparison to the normal individuals. Also, six protein spots were confirmed by western-blot analysis. In conclusion, all the twenty-four identified proteins are involved in pathways which their up- or down-regulation leads to the accumulation of undesired substrates, cell death and apoptosis. Bioinformatics' tools indicated that these identified proteins were involved in various metabolic processes, DNA damage response, immune response and etc. The present study provides a suitable platform for further clinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

PubMed

Nanduri, Bindu; Shack, Leslie A; Rai, Aswathy N; Epperson, William B; Baumgartner, Wes; Schmidt, Ty B; Edelmann, Mariola J

2016-12-15

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics. Copyright © 2016 Elsevier Inc. All rights reserved.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

PubMed Central

Saylor, Karen L.; Anver, Miriam R.; Salomon, David S.; Golubeva, Yelena G.

2016-01-01

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM. PMID:27077656

Rapid Assessment of Contaminants and Interferences in Mass Spectrometry Data Using Skyline

NASA Astrophysics Data System (ADS)

Rardin, Matthew J.

2018-04-01

Proper sample preparation in proteomic workflows is essential to the success of modern mass spectrometry experiments. Complex workflows often require reagents which are incompatible with MS analysis (e.g., detergents) necessitating a variety of sample cleanup procedures. Efforts to understand and mitigate sample contamination are a continual source of disruption with respect to both time and resources. To improve the ability to rapidly assess sample contamination from a diverse array of sources, I developed a molecular library in Skyline for rapid extraction of contaminant precursor signals using MS1 filtering. This contaminant template library is easily managed and can be modified for a diverse array of mass spectrometry sample preparation workflows. Utilization of this template allows rapid assessment of sample integrity and indicates potential sources of contamination. [Figure not available: see fulltext.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

PubMed

Calderón-Celis, Francisco; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo

2017-07-31

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review. © 2017 Wiley Periodicals, Inc.

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

PubMed Central

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A.

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding. PMID:25870605

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

PubMed

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

Microscale immobilized enzyme reactors in proteomics: latest developments.

PubMed

Safdar, Muhammad; Spross, Jens; Jänis, Janne

2014-01-10

Enzymatic digestion of proteins is one of the key steps in proteomic analyses. There has been a steady progress in the applied digestion protocols in the past, starting from conventional time-consuming in-solution or in-gel digestion protocols to rapid and efficient methods utilizing different types of microscale enzyme reactors. Application of such microreactors has been proven beneficial due to lower sample consumption, higher sensitivity and straightforward coupling with LC-MS set-ups. Novel stationary phases, immobilization techniques and device formats are being constantly developed and tested to optimize digestion efficiency of proteolytic enzymes. This review focuses on the latest developments associated with the preparation and application of microscale enzyme reactors for proteomics applications since 2008 onwards. A special attention has been paid to the discussion of different stationary phases applied for immobilization purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.

PubMed

Planatscher, Hannes; Supper, Jochen; Poetz, Oliver; Stoll, Dieter; Joos, Thomas; Templin, Markus F; Zell, Andreas

2010-06-25

Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

Development of Advanced Technologies for Complete Genomic and Proteomic Characterization of Quantized Human Tumor Cells

DTIC Science & Technology

2013-07-01

AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT With the establishment of GBM cell lines...and quantized cell populations form these GBM patients tumor samples we are able to complete some of our aims of our project. We will continue to...collect tumor samples with consent from families of GBM patients in preparation to perform the molecular analysis of these. Our efforts in the development

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines

PubMed Central

Togayachi, Akira; Azadi, Parastoo; Ishihara, Mayumi; Geyer, Rudolf; Galuska, Christina; Geyer, Hildegard; Kakehi, Kazuaki; Kinoshita, Mitsuhiro; Karlsson, Niclas G.; Jin, Chunsheng; Kato, Koichi; Yagi, Hirokazu; Kondo, Sachiko; Kawasaki, Nana; Hashii, Noritaka; Kolarich, Daniel; Stavenhagen, Kathrin; Packer, Nicolle H.; Thaysen-Andersen, Morten; Nakano, Miyako; Taniguchi, Naoyuki; Kurimoto, Ayako; Wada, Yoshinao; Tajiri, Michiko; Yang, Pengyuan; Cao, Weiqian; Li, Hong; Rudd, Pauline M.; Narimatsu, Hisashi

2016-01-01

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples. PMID:26511985

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

PubMed

Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

2016-01-05

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Plant membrane proteomics.

PubMed

Ephritikhine, Geneviève; Ferro, Myriam; Rolland, Norbert

2004-12-01

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.

Proteoglycomics: Recent Progress and Future Challenges

PubMed Central

Ly, Mellisa; Laremore, Tatiana N.

2010-01-01

Abstract Proteoglycomics is a systematic study of structure, expression, and function of proteoglycans, a posttranslationally modified subset of a proteome. Although relying on the established technologies of proteomics and glycomics, proteoglycomics research requires unique approaches for elucidating structure–function relationships of both proteoglycan components, glycosaminoglycan chain, and core protein. This review discusses our current understanding of structure and function of proteoglycans, major players in the development, normal physiology, and disease. A brief outline of the proteoglycomic sample preparation and analysis is provided along with examples of several recent proteoglycomic studies. Unique challenges in the characterization of glycosaminoglycan component of proteoglycans are discussed, with emphasis on the many analytical tools used and the types of information they provide. PMID:20450439

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance*

PubMed Central

Paulovich, Amanda G.; Billheimer, Dean; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Tabb, David L.; Wang, Pei; Blackman, Ronald K.; Bunk, David M.; Cardasis, Helene L.; Clauser, Karl R.; Kinsinger, Christopher R.; Schilling, Birgit; Tegeler, Tony J.; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fenyo, David; Carr, Steven A.; Fisher, Susan J.; Gibson, Bradford W.; Mesri, Mehdi; Neubert, Thomas A.; Regnier, Fred E.; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E.; Tempst, Paul; Liebler, Daniel C.

2010-01-01

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. PMID:19858499

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Tools for phospho- and glycoproteomics of plasma membranes.

PubMed

Wiśniewski, Jacek R

2011-07-01

Analysis of plasma membrane proteins and their posttranslational modifications is considered as important for identification of disease markers and targets for drug treatment. Due to their insolubility in water, studying of plasma membrane proteins using mass spectrometry has been difficult for a long time. Recent technological developments in sample preparation together with important improvements in mass spectrometric analysis have facilitated analysis of these proteins and their posttranslational modifications. Now, large scale proteomic analyses allow identification of thousands of membrane proteins from minute amounts of sample. Optimized protocols for affinity enrichment of phosphorylated and glycosylated peptides have set new dimensions in the depth of characterization of these posttranslational modifications of plasma membrane proteins. Here, I summarize recent advances in proteomic technology for the characterization of the cell surface proteins and their modifications. In the focus are approaches allowing large scale mapping rather than analytical methods suitable for studying individual proteins or non-complex mixtures.

The heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous analysis of proteomes and transcriptomes.

PubMed

Wagner, Wolfgang; Feldmann, Robert E; Seckinger, Anja; Maurer, Martin H; Wein, Frederik; Blake, Jonathon; Krause, Ulf; Kalenka, Armin; Bürgers, Heinrich F; Saffrich, Rainer; Wuchter, Patrick; Kuschinsky, Wolfgang; Ho, Anthony D

2006-04-01

Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations. In our continuing effort to characterize MSC, we have analyzed the differential transcriptome and proteome expression profiles of MSC preparations isolated from human bone marrow under two different expansion media (BM-MSC-M1 and BM-MSC-M2). In proteomics, 136 protein spots were unambiguously identified by MALDI-TOF-MS and corresponding cDNA spots were selected on our "Human Transcriptome cDNA Microarray." Combination of datasets revealed a correlation in differential gene expression and protein expression of BM-MSC-M1 vs BM-MSC-M2. Genes involved in metabolism were more highly expressed in BM-MSC-M1, whereas genes involved in development, morphogenesis, extracellular matrix, and differentiation were more highly expressed in BM-MSC-M2. Interchanging culture conditions for 8 days revealed that differential expression was retained in several genes whereas it was altered in others. Our results have provided evidence that homogeneous BM-MSC preparations can reproducibly be isolated under standardized conditions, whereas culture conditions exert a prominent impact on transcriptome, proteome, and cellular organization of BM-MSC.

Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis

PubMed Central

Macdonald, Lucy J.; Adams, Paul D.; Petzold, Christopher J.; Strabala, Timothy J.; Wagner, Armin; Heazlewood, Joshua L.

2013-01-01

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557. PMID:24416096

Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics.

PubMed

Ni, Mao-Wei; Wang, Lu; Chen, Wei; Mou, Han-Zhou; Zhou, Jie; Zheng, Zhi-Guo

2017-01-30

Mass spectrometry (MS)-based protein identification depends mainly on protein extraction and digestion. Although sodium dodecyl sulfate (SDS) can preclude enzymatic digestion and interfere with MS analysis, it is still the most widely used surfactant in these steps. To overcome these disadvantages, a SDS-compatible proteomic technique for SDS removal prior to MS-based analyses was developed, namely filter-aided sample preparation (FASP). Herein, based on the effectiveness of sodium deoxycholate and a detergent removal spin column, we developed a modified FASP (mFASP) method and compared its overall performance, total number of peptides and proteins identified for shotgun proteomic experiments with that of the FASP method. Identification of 4570 ± 392 and 9139 ± 317 peptides and description of 862 ± 46 and 1377 ± 33 protein groups with two or more peptides from the ovarian cancer cell line A2780 was accomplished by FASP and mFASP methods, respectively. The mFASP method (21.2 ± 0.2%) had higher average peptide to protein coverage than FASP method (13.2 ± 0.5%). More hydrophobic peptides were identified by mFASP than by FASP, as indicated by the GRAVY score distribution. The reported method enables reliable and efficient identification of proteins and peptides in whole-cell extracts containing SDS. The new approach allows for higher throughput (the simultaneous identification of more proteins), a more comprehensive investigation of proteins, and potentially the discovery of new biomarkers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Proteomics Analysis of Tissue Samples Reveals Changes in Mitochondrial Protein Levels in Parathyroid Hyperplasia over Adenoma

PubMed Central

AKPINAR, GURLER; KASAP, MURAT; CANTURK, NUH ZAFER; ZULFIGAROVA, MEHIN; ISLEK, EYLÜL ECE; GULER, SERTAC ATA; SIMSEK, TURGAY; CANTURK, ZEYNEP

2017-01-01

Background/Aim: To unveil the pathophysiology of primary hyperparathyroidism, molecular details of parathyroid hyperplasia and adenoma have to be revealed. Such details will provide the tools necessary for differentiation of these two look-alike diseases. Therefore, in the present study, a comparative proteomic study using postoperative tissue samples from the parathyroid adenoma and parathyroid hyperplasia patients was performed. Materials and Methods: Protein extracts were prepared from tissue samples (n=8 per group). Protein pools were created for each group and subjected to DIGE and conventional 2DE. Following image analysis, spots representing the differentially regulated proteins were excised from the and used for identification via MALDI-TOF/TOF analysis. Results: The identities of 40 differentially-expressed proteins were revealed. Fourteen of these proteins were over-expressed in the hyperplasia while 26 of them were over-expressed in the adenoma. Conclusion: Most proteins found to be over-expressed in the hyperplasia samples were mitochondrial, underlying the importance of the mitochondrial activity as a potential biomarker for differentiation of parathyroid hyperplasia from adenoma. PMID:28446534

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen*

PubMed Central

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

Bacteriophage cell lysis of Shiga toxin-producing Escherichia coli for top-down proteomic identification of Shiga toxin 1 & 2 using matrix-assisted laser desorption/ionization tandem time-of-light mass spectrometry

USDA-ARS?s Scientific Manuscript database

RATIONALE: Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage...

Review of sample preparation strategies for MS-based metabolomic studies in industrial biotechnology.

PubMed

Causon, Tim J; Hann, Stephan

2016-09-28

Fermentation and cell culture biotechnology in the form of so-called "cell factories" now play an increasingly significant role in production of both large (e.g. proteins, biopharmaceuticals) and small organic molecules for a wide variety of applications. However, associated metabolic engineering optimisation processes relying on genetic modification of organisms used in cell factories, or alteration of production conditions remain a challenging undertaking for improving the final yield and quality of cell factory products. In addition to genomic, transcriptomic and proteomic workflows, analytical metabolomics continues to play a critical role in studying detailed aspects of critical pathways (e.g. via targeted quantification of metabolites), identification of biosynthetic intermediates, and also for phenotype differentiation and the elucidation of previously unknown pathways (e.g. via non-targeted strategies). However, the diversity of primary and secondary metabolites and the broad concentration ranges encompassed during typical biotechnological processes means that simultaneous extraction and robust analytical determination of all parts of interest of the metabolome is effectively impossible. As the integration of metabolome data with transcriptome and proteome data is an essential goal of both targeted and non-targeted methods addressing production optimisation goals, additional sample preparation steps beyond necessary sampling, quenching and extraction protocols including clean-up, analyte enrichment, and derivatisation are important considerations for some classes of metabolites, especially those present in low concentrations or exhibiting poor stability. This contribution critically assesses the potential of current sample preparation strategies applied in metabolomic studies of industrially-relevant cell factory organisms using mass spectrometry-based platforms primarily coupled to liquid-phase sample introduction (i.e. flow injection, liquid chromatography, or capillary electrophoresis). Particular focus is placed on the selectivity and degree of enrichment attainable, as well as demands of speed, absolute quantification, robustness and, ultimately, consideration of fully-integrated bioanalytical solutions to optimise sample handling and throughput. Copyright © 2016 Elsevier B.V. All rights reserved.

Deep coverage of the beer proteome.

PubMed

Grochalová, Martina; Konečná, Hana; Stejskal, Karel; Potěšil, David; Fridrichová, Danuše; Srbová, Eva; Ornerová, Kateřina; Zdráhal, Zbyněk

2017-06-06

We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. The study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves.

PubMed

Kulkarni, Ketav P; Ramarathinam, Sri H; Friend, James; Yeo, Leslie; Purcell, Anthony W; Perlmutter, Patrick

2010-06-21

A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: Systematic variation between sample types and calibration of mass spectrometry data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haab, Brian B.; Geierstanger, Bernhard H.; Michailidis, George

2005-08-01

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets usingmore » the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.« less

Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

PubMed Central

Wang, Xiaxia; Zhao, Xiaowei; Huang, Dongwei; Pan, Xiaocheng; Qi, Yunxia; Yang, Yongxin; Zhao, Huiling; Cheng, Guanglong

2017-01-01

Casein micelles contribute to the physicochemical properties of milk and may also influence its functionality. At present, however, there is an incomplete understanding of the casein micelle associated proteins and its diversity among the milk obtained from different species. Therefore, milk samples were collected from seven dairy animals groups, casein fractions were prepared by ultracentrifugation and their constituent proteins were identified by liquid chromatography tandem mass spectrometry. A total of 193 distinct proteins were identified among all the casein micelle preparations. Protein interaction analysis indicated that caseins could interact with major whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, and serum albumin, and then whey proteins interacted with other proteins. Pathway analysis found that the peroxisome proliferator-activated receptor signaling pathway is shared among the studied animals. Additionally, galactose metabolism pathway is also found to be commonly involved for proteins derived from camel and horse milk. According to the similarity of casein micelle proteomes, two major sample clusters were classified into ruminant animals (Holstein and Jersey cows, buffaloes, yaks, and goats) and non-ruminants (camels and horses). Our results provide new insights into the protein profile associated with casein micelles and the functionality of the casein micelle from the studied animals. PMID:28240229

Identification of Phosphorylated Proteins on a Global Scale.

PubMed

Iliuk, Anton

2018-05-31

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Lysine acetylation stoichiometry and proteomics analyses reveal pathways regulated by sirtuin 1 in human cells.

PubMed

Gil, Jeovanis; Ramírez-Torres, Alberto; Chiappe, Diego; Luna-Peñaloza, Juan; Fernandez-Reyes, Francis C; Arcos-Encarnación, Bolivar; Contreras, Sandra; Encarnación-Guevara, Sergio

2017-11-03

Lysine acetylation is a widespread posttranslational modification affecting many biological pathways. Recent studies indicate that acetylated lysine residues mainly exhibit low acetylation occupancy, but challenges in sample preparation and analysis make it difficult to confidently assign these numbers, limiting understanding of their biological significance. Here, we tested three common sample preparation methods to determine their suitability for assessing acetylation stoichiometry in three human cell lines, identifying the acetylation occupancy in more than 1,300 proteins from each cell line. The stoichiometric analysis in combination with quantitative proteomics also enabled us to explore their functional roles. We found that higher abundance of the deacetylase sirtuin 1 (SIRT1) correlated with lower acetylation occupancy and lower levels of ribosomal proteins, including those involved in ribosome biogenesis and rRNA processing. Treatment with the SIRT1 inhibitor EX-527 confirmed SIRT1's role in the regulation of pre-rRNA synthesis and processing. Specifically, proteins involved in pre-rRNA transcription, including subunits of the polymerase I and SL1 complexes and the RNA polymerase I-specific transcription initiation factor RRN3, were up-regulated after SIRT1 inhibition. Moreover, many protein effectors and regulators of pre-rRNA processing needed for rRNA maturation were also up-regulated after EX-527 treatment with the outcome that pre-rRNA and 28S rRNA levels also increased. More generally, we found that SIRT1 inhibition down-regulates metabolic pathways, including glycolysis and pyruvate metabolism. Together, these results provide the largest data set thus far of lysine acetylation stoichiometry (available via ProteomeXchange with identifier PXD005903) and set the stage for further biological investigations of this central posttranslational modification. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

RaftProt: mammalian lipid raft proteome database.

PubMed

Shah, Anup; Chen, David; Boda, Akash R; Foster, Leonard J; Davis, Melissa J; Hill, Michelle M

2015-01-01

RaftProt (http://lipid-raft-database.di.uq.edu.au/) is a database of mammalian lipid raft-associated proteins as reported in high-throughput mass spectrometry studies. Lipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids thought to act as dynamic signalling and sorting platforms. Given their fundamental roles in cellular regulation, there is a plethora of information on the size, composition and regulation of these membrane microdomains, including a large number of proteomics studies. To facilitate the mining and analysis of published lipid raft proteomics studies, we have developed a searchable database RaftProt. In addition to browsing the studies, performing basic queries by protein and gene names, searching experiments by cell, tissue and organisms; we have implemented several advanced features to facilitate data mining. To address the issue of potential bias due to biochemical preparation procedures used, we have captured the lipid raft preparation methods and implemented advanced search option for methodology and sample treatment conditions, such as cholesterol depletion. Furthermore, we have identified a list of high confidence proteins, and enabled searching only from this list of likely bona fide lipid raft proteins. Given the apparent biological importance of lipid raft and their associated proteins, this database would constitute a key resource for the scientific community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Proteomic profile of serum of pregnant women carring a fetus with Down syndrome using nano uplc Q-tof ms/ms technology.

PubMed

López Uriarte, Graciela Arelí; Burciaga Flores, Carlos Horacio; Torres de la Cruz, Víctor Manuel; Medina Aguado, María Magdalena; Gómez Puente, Viviana Maricela; Romero Gutiérrez, Liliana Nayeli; Martínez de Villarreal, Laura Elia

2018-06-01

Prenatal diagnosis of Down syndrome (DS) is based on the calculated risk of maternal age, biochemical and ultrasonographic markers and recently by cfDNA. Differences in proteomic profiles may give an opportunity to find new biomarkers. Characterize proteome of serum of mothers carrying DS fetus. Blood serum samples of three groups of women were obtained, (a) 10 non-pregnant, (b) 10 pregnant with healthy fetus by ultrasound evaluation, (c) nine pregnant with DS fetus. Sample preparation was as follows: Albumin/IgG depletion, desalting, and trypsin digestion; the process was performed in nanoUPLC MS/MS. Data analysis was made with Mass Lynx 4.1 and ProteinLynx Global Server 3.0, peptide and protein recognition by MASCOT algorithm and UNIPROT-Swissprot database. Each group showed different protein profiles. Some proteins were shared between groups. Only sera from pregnant women showed proteins related to immune and clot pathways. Mothers with DS fetus had 42 specific proteins. We found a different serum protein profile in mothers carrying DS fetuses that do not reflect expression of genes in the extra chromosome. Further studies will be necessary to establish the role of these proteins in aneuploid fetus and analyze their possible use as potential biomarkers.

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

PubMed

Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

2016-09-16

The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Missing Proteins Defined by Chromosome-Centric Proteome Project in the Cytoplasmic Detergent-Insoluble Proteins.

PubMed

Chen, Yang; Li, Yaxing; Zhong, Jiayong; Zhang, Jing; Chen, Zhipeng; Yang, Lijuan; Cao, Xin; He, Qing-Yu; Zhang, Gong; Wang, Tong

2015-09-04

Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2-4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins. We optimized a workflow and separated cytoplasmic DIPs from three human lung and three human hepatoma cell lines via differential speed centrifugation. We verified that the detergent-soluble proteins (DSPs) could be sufficiently depleted and the cytoplasmic DIP isolation was partially reproducible with Spearman r > 0.70 according to two independent SILAC MS experiments. Through label-free MS, we identified 4524 and 4156 DIPs from lung and liver cells, respectively. Among them, a total of 23 missing proteins (22 PE2 and 1 PE4) were identified by MS, and 18 of them had translation evidence; in addition, six PE5 proteins were identified by MS, three with translation evidence. We showed that cytoplasmic DIPs were not an enrichment of transmembrane proteins and were chromosome-, cell type-, and tissue-specific. Furthermore, we demonstrated that DIPs were distinct from DSPs in terms of structural and physical-chemical features. In conclusion, we have found 23 missing proteins and 6 PE5 proteins from the cytoplasmic insoluble proteome that is biologically and physical-chemically different from the soluble proteome, suggesting that cytoplasmic DIPs carry comprehensive and valuable information for finding PE of missing proteins. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001694.

Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry

PubMed Central

Krey, Jocelyn F.; Wilmarth, Philip A.; David, Larry L.; Barr-Gillespie, Peter G.

2017-01-01

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle-protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel-reaction-monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type and mice with deafness mutations affecting hair-bundle proteins. (120 words; maximum 250) PMID:28109437

Ultrasonic-based membrane aided sample preparation of urine proteomes.

PubMed

Jesus, Jemmyson Romário; Santos, Hugo M; López-Fernández, H; Lodeiro, Carlos; Arruda, Marco Aurélio Zezzi; Capelo, J L

2018-02-01

A new ultrafast ultrasonic-based method for shotgun proteomics as well as label-free protein quantification in urine samples is developed. The method first separates the urine proteins using nitrocellulose-based membranes and then proteins are in-membrane digested using trypsin. The enzymatic digestion process is accelerated from overnight to four minutes using a sonoreactor ultrasonic device. Overall, the sample treatment pipeline comprising protein separation, digestion and identification is done in just 3h. The process is assessed using urine of healthy volunteers. The method shows that male can be differentiated from female using the protein content of urine in a fast, easy and straightforward way. 232 and 226 proteins are identified in urine of male and female, respectively. From this, 162 are common to both genders, whilst 70 are unique to male and 64 to female. From the 162 common proteins, 13 are present at levels statistically different (p < 0.05). The method matches the analytical minimalism concept as outlined by Halls, as each stage of this analysis is evaluated to minimize the time, cost, sample requirement, reagent consumption, energy requirements and production of waste products. Copyright © 2017 Elsevier B.V. All rights reserved.

Microfluidics for the analysis of membrane proteins: how do we get there?

PubMed

Battle, Katrina N; Uba, Franklin I; Soper, Steven A

2014-08-01

The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and fractionation of membranes for proteomic analyses.

PubMed

Marmagne, Anne; Salvi, Daniel; Rolland, Norbert; Ephritikhine, Geneviève; Joyard, Jacques; Barbier-Brygoo, Hélène

2006-01-01

Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.

Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

PubMed Central

Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

2016-01-01

Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis – An update covering the period 2011-2014

PubMed Central

Puangpila, Chanida; Mayadunne, Erandi; Rassi, Ziad El

2015-01-01

This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

PubMed

Molina, Laurence; Salvetat, Nicolas; Ameur, Randa Ben; Peres, Sabine; Sommerer, Nicolas; Jarraya, Fayçal; Ayadi, Hammadi; Molina, Franck; Granier, Claude

2011-12-10

The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual. Copyright © 2011 Elsevier B.V. All rights reserved.

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

PubMed Central

2013-01-01

Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx). PMID:23758935

NCI Blog Post: CPTAC, the Complementary Sibling of TCGA (An Interview with Dr. Henry Rodriguez about NCI’s Proteomics Program) | Office of Cancer Clinical Proteomics Research

Cancer.gov

What is proteomics? Proteomics is a highly automated and rapid method for measuring all the proteins in a biological sample. Proteins are the molecules that actually do most of the work inside a cell. When researchers develop cancer drugs, those drugs typically target proteins, so scientists and clinicians really have to understand what the proteins are doing. Proteomics researchers are now able to measure up to 10,000 proteins per tumor sample.

Proteome and Antigens of Mycobacterium avium subspecies Paratuberculosis

USDA-ARS?s Scientific Manuscript database

This chapter will review what is known about the proteome of this significant veterinary pathogen by discussing the use of various whole cell preparations and fractionated components thereof. With improvements in technology, many new modifications to the traditional ELISA assay have been introduced...

Comparative proteomic analysis of pituitary glands from Huoyan geese between pre-laying and laying periods using an iTRAQ-based approach

PubMed Central

Xing, Zhe; Liu, Mei; Gao, Ming; Meng, Bo; Fan, Ruiming

2017-01-01

In this study, we performed a comprehensive evaluation of the proteomic profile of the pituitary gland of the Huoyan goose during the laying period compared to the pre-laying period using an iTRAQ-based approach. Protein samples were prepared from pituitary gland tissues of nine pre-laying period and nine laying period geese. Then the protein samples from three randomly selected geese within each period were pooled in equal amounts to generate one biological sample pool. We identified 684 differentially expressed proteins, including 418 up-regulated and 266 down-regulated proteins. GO annotation and KEGG pathway analyses of these proteins were conducted. Some of these proteins were found to be associated with hormone and neurotransmitter secretion and transport, neuropeptide signalling and GnRH signalling pathways, among others. Subsequently, the modification of the abundance of three proteins (prolactin, chromogranin-A and ITPR3) was verified using western blotting. Our results will provide a new source for mining genes and gene products related to the egg-laying performance of Huoyan geese, and may provide important information for the conservation and utilization of local goose breeds. PMID:28945779

HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages.

PubMed

Wang, Jing; Gu, Yong; Wang, Lihong; Hang, Xingyi; Gao, Yan; Wang, Hangyan; Zhang, Chenggang

2007-11-01

This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.

Milk Bottom-Up Proteomics: Method Optimization

PubMed Central

Vincent, Delphine; Ezernieks, Vilnis; Elkins, Aaron; Nguyen, Nga; Moate, Peter J.; Cocks, Benjamin G.; Rochfort, Simone

2016-01-01

Milk is a complex fluid whose proteome displays a diverse set of proteins of high abundance such as caseins and medium to low abundance whey proteins such as ß-lactoglobulin, lactoferrin, immunoglobulins, glycoproteins, peptide hormones, and enzymes. A sample preparation method that enables high reproducibility and throughput is key in reliably identifying proteins present or proteins responding to conditions such as a diet, health or genetics. Using skim milk samples from Jersey and Holstein-Friesian cows, we compared three extraction procedures which have not previously been applied to samples of cows' milk. Method A (urea) involved a simple dilution of the milk in a urea-based buffer, method B (TCA/acetone) involved a trichloroacetic acid (TCA)/acetone precipitation, and method C (methanol/chloroform) involved a tri-phasic partition method in chloroform/methanol solution. Protein assays, SDS-PAGE profiling, and trypsin digestion followed by nanoHPLC-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analyses were performed to assess their efficiency. Replicates were used at each analytical step (extraction, digestion, injection) to assess reproducibility. Mass spectrometry (MS) data are available via ProteomeXchange with identifier PXD002529. Overall 186 unique accessions, major and minor proteins, were identified with a combination of methods. Method C (methanol/chloroform) yielded the best resolved SDS-patterns and highest protein recovery rates, method A (urea) yielded the greatest number of accessions, and, of the three procedures, method B (TCA/acetone) was the least compatible of all with a wide range of downstream analytical procedures. Our results also highlighted breed differences between the proteins in milk of Jersey and Holstein-Friesian cows. PMID:26793233

Comparative and Quantitative Global Proteomics Approaches: An Overview

PubMed Central

Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis

2013-01-01

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403

CPTAC Releases Cancer Proteome Confirmatory Colon Study Data | Office of Cancer Clinical Proteomics Research

Cancer.gov

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) announces the release of the cancer proteome confirmatory colon study data. The goal of the study is to analyze the proteomes of approximately 100 confirmatory colon tumor patients, which includes tumor and adjacent normal samples, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) global proteomic and phosphoproteomic profiling.

A rapid method for preparation of the cerebrospinal fluid proteome.

PubMed

Larssen, Eivind; Brede, Cato; Hjelle, Anne Bjørnstad; Øysaed, Kjell Birger; Tjensvoll, Anne Bolette; Omdal, Roald; Ruoff, Peter

2015-01-01

The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high-abundance proteins (HAPs) can interfere with the detection of low-abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Adaptations to Starvation Prepare Escherichia coli for Disinfection Tolerance

PubMed Central

Du, Zhe; Nandakumar, Renu; Nickerson, Kenneth; Li, Xu

2015-01-01

Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms. PMID:25463932

Proteomic adaptations to starvation prepare Escherichia coli for disinfection tolerance.

PubMed

Du, Zhe; Nandakumar, Renu; Nickerson, Kenneth W; Li, Xu

2015-02-01

Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification.

PubMed

Hains, Peter G; Robinson, Phillip J

2017-09-01

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2-5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics.

Mass spectrometry-based proteomics for translational research: a technical overview.

PubMed

Paulo, Joao A; Kadiyala, Vivek; Banks, Peter A; Steen, Hanno; Conwell, Darwin L

2012-03-01

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.

Mass Spectrometry-Based Proteomics for Translational Research: A Technical Overview

PubMed Central

Paulo, Joao A.; Kadiyala, Vivek; Banks, Peter A.; Steen, Hanno; Conwell, Darwin L.

2012-01-01

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease. PMID:22461744

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome.

PubMed

Pieper, Rembert; Su, Qin; Gatlin, Christine L; Huang, Shih-Ting; Anderson, N Leigh; Steiner, Sandra

2003-04-01

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.

Evaluation of SDS depletion using an affinity spin column and IMS-MS detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hengel, Shawna M.; Floyd, Erica A.; Baker, Erin Shammel

2012-11-01

While the use of detergents is necessary for a variety of protein isolation preparation protocols, often prior to mass spectral (MS) analysis, they are not compatible with MS analysis due to ion suppression and adduct formation. This manuscript describes optimization of detergent removal, using commercially available SDS depletion spin columns containing an affinity resin, providing for both increased protein recovery and thorough SDS removal. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) allowed for a concurrent analysis of both analyte and detergent. In the case of both proteins and peptides, higher detergent concentrations than previously reported provided an increase ofmore » sample recovery; however there was a limit as SDS was detected by IMS-MS at higher levels of SDS indicating incomplete detergent depletion. The results also suggest optimal conditions for SDS removal are dependent on the sample concentration. Overall, this study provides a useful guide for proteomic studies where SDS is required for efficient sample preparation.« less

Proteomic and peptidomic analysis of human sweat with emphasis on proteolysis.

PubMed

Yu, Yijing; Prassas, Ioannis; Muytjens, Carla M J; Diamandis, Eleftherios P

2017-02-23

Sweat is produced by eccrine and apocrine glands and represents a biological fluid with established roles in thermo-regulation and host infection defense. The composition of sweat is highly dynamic and alters significantly in various skin and other disorders. Therefore, in-depth profiling of sweat protein composition is expected to augment our understanding of the pathobiology of several skin diseases and may lead to the identification of useful sweat-based disease biomarkers. We here reported an in-depth analysis of the human sweat proteome and peptidome. Sweat was collected from healthy males and healthy females of ages 20-60years, following strenuous exercise. Two sweat pools were prepared (1 for males and 1 for females) and were subjected to sample preparation for mass spectrometric analysis. We identified a total of 861 unique proteins during our proteomic analysis and 32,818 endogenous peptides (corresponding to additional 1067 proteins) from our peptidomics workflow. As expected, the human skin was identified as the most abundant source of sweat proteins and peptides. Several skin proteases and protease inhibitors were identified in human sweat, highlighting the intense proteolytic activity of human skin. The presence of several antimicrobial peptides supports the functional roles of sweat in host defense and innate immunity. Sweat is a skin-associated biological fluid, secreted by eccrine and apocrine glands, with essential function in body thermo-regulation and host infection defense. In the present study, we performed in-depth profiling of both sweat proteome and peptidome composition. Our data provide the most in-depth characterization of the skin's catalytic network present in sweat. For the first time, we brought to light novel peptides in human sweat that potentially have antimicrobial activity, which highlight the important role of this fluid in innate immunity. All these findings allow us to have a better understanding of the complex web of proteases in skin and may act as a platform for the future discovery of novel skin biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomic Analysis to Identify Functional Molecules in Drug Resistance Caused by E-Cadherin Knockdown in 3D-Cultured Colorectal Cancer Models

DTIC Science & Technology

2014-09-01

total number of 538 phosphopeptides were identified, among which 350 phosphopeptides had been identified with the first round of TiO2 enrichment and 430...year research and the collection of proteomic and phosphoproteomic data is still in process. PRODUCTS Manuscripts: Yue XS , Hummon AB. Combining...of IMAC and TiO2 enrichment methods to increase phosphoproteomic identifications, manuscript in preparation. Yue XS , Hummon AB. Proteomic and

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice.

PubMed

Osteikoetxea, Xabier; Benke, Márton; Rodriguez, Marta; Pálóczi, Krisztina; Sódar, Barbara W; Szvicsek, Zsuzsanna; Szabó-Taylor, Katalin; Vukman, Krisztina V; Kittel, Ágnes; Wiener, Zoltán; Vékey, Károly; Harsányi, László; Szűcs, Ákos; Turiák, Lilla; Buzás, Edit I

2018-04-30

The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. Comparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Metaproteomics as a Complementary Approach to Gut Microbiota in Health and Disease

NASA Astrophysics Data System (ADS)

Petriz, Bernardo A.; Franco, Octávio L.

2017-01-01

Classic studies on phylotype profiling are limited to the identification of microbial constituents, where information is lacking about the molecular interaction of these bacterial communities with the host genome and the possible outcomes in host biology. A range of OMICs approaches have provided great progress linking the microbiota to health and disease. However, the investigation of this context through proteomic mass spectrometry-based tools is still being improved. Therefore, metaproteomics or community proteogenomics has emerged as a complementary approach to metagenomic data, as a field in proteomics aiming to perform large-scale characterization of proteins from environmental microbiota such as the human gut. The advances in molecular separation methods coupled with mass spectrometry (e.g. LC-MS/MS) and proteome bioinformatics have been fundamental in these novel large-scale metaproteomic studies, which have further been performed in a wide range of samples including soil, plant and human environments. Metaproteomic studies will make major progress if a comprehensive database covering the genes and expresses proteins from all gut microbial species is developed. To this end, we here present some of the main limitations of metaproteomic studies in complex microbiota environments such as the gut, also addressing the up-to-date pipelines in sample preparation prior to fractionation/separation and mass spectrometry analysis. In addition, a novel approach to the limitations of metagenomic databases is also discussed. Finally, prospects are addressed regarding the application of metaproteomic analysis using a unified host-microbiome gene database and other meta-OMICs platforms.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

PubMed Central

Samardzija, Chantel; Greening, David W.; Escalona, Ruth; Chen, Maoshan; Bilandzic, Maree; Luwor, Rodney; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat

2017-01-01

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. PMID:28406185

CPTAC researchers report first large-scale integrated proteomic and genomic analysis of a human cancer | Office of Cancer Clinical Proteomics Research

Cancer.gov

Investigators from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) who comprehensively analyzed 95 human colorectal tumor samples, have determined how gene alterations identified in previous analyses of the same samples are expressed at the protein level. The integration of proteomic and genomic data, or proteogenomics, provides a more comprehensive view of the biological features that drive cancer than genomic analysis alone and may help identify the most important targets for cancer detection and intervention.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

PubMed Central

Szymanski, Witold G.; Kierszniowska, Sylwia; Schulze, Waltraud X.

2013-01-01

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4. PMID:24121251

CPTAC Prospective Biospecimen Collection Solicitation | Office of Cancer Clinical Proteomics Research

Cancer.gov

A new funding opportunity in support of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) seeks to prospectively procure tumor samples, collected for proteomics investigation.

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease*

PubMed Central

Schnell, Gilles; Boeuf, Amandine; Westermann, Benoît; Jaulhac, Benoît; Lipsker, Dan; Carapito, Christine; Boulanger, Nathalie; Ehret-Sabatier, Laurence

2015-01-01

Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy). PMID:25713121

Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Ying; Zhao, Rui; Piehowski, Paul D.

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading tomore » a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.« less

Proteomic diversity of high-density lipoprotein explains its association with clinical outcome in patients with heart failure.

PubMed

Emmens, Johanna Elisabeth; Jones, Donald J L; Cao, Thong H; Chan, Daniel C S; Romaine, Simon P R; Quinn, Paulene A; Anker, Stefan D; Cleland, John G; Dickstein, Kenneth; Filippatos, Gerasimos; Hillege, Hans L; Lang, Chim C; Ponikowski, Piotr; Samani, Nilesh J; van Veldhuisen, Dirk J; Zannad, Faiz; Zwinderman, Aeilko H; Metra, Marco; de Boer, Rudolf A; Voors, Adriaan A; Ng, Leong L

2018-02-01

Previously, low high-density lipoprotein (HDL) cholesterol was found to be one of the strongest predictors of mortality and/or heart failure (HF) hospitalisation in patients with HF. We therefore performed in-depth investigation of the multifunctional HDL proteome to reveal underlying pathophysiological mechanisms explaining the association between HDL and clinical outcome. We selected a cohort of 90 HF patients with 1:1 cardiovascular death/survivor ratio from BIOSTAT-CHF. A novel optimised protocol for selective enrichment of lipoproteins was used to prepare plasma. Enriched lipoprotein content of samples was analysed using high resolution nanoscale liquid chromatography-mass spectrometry-based proteomics, utilising a label free approach. Within the HDL proteome, 49 proteins significantly differed between deaths and survivors. An optimised model of 12 proteins predicted death with 76% accuracy (Nagelkerke R 2 =0.37, P < 0.001). The strongest contributors to this model were filamin-A (related to crosslinking of actin filaments) [odds ratio (OR) 0.31, 95% confidence interval (CI) 0.15-0.61, P = 0.001] and pulmonary surfactant-associated protein B (related to alveolar capillary membrane function) (OR 2.50, 95% CI 1.57-3.98, P < 0.001). The model predicted mortality with an area under the curve of 0.82 (95% CI 0.77-0.87, P < 0.001). Internal cross validation resulted in 73.3 ± 7.2% accuracy. This study shows marked differences in composition of the HDL proteome between HF survivors and deaths. The strongest differences were seen in proteins reflecting crosslinking of actin filaments and alveolar capillary membrane function, posing potential pathophysiological mechanisms underlying the association between HDL and clinical outcome in HF. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

CPTAC Releases Largest-Ever Breast Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have released a dataset of proteins and  phosphopeptides identified through deep proteomic and phosphoproteomic analysis of breast tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).

The application of proteomics in different aspects of hepatocellular carcinoma research.

PubMed

Xing, Xiaohua; Liang, Dong; Huang, Yao; Zeng, Yongyi; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng

2016-08-11

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which is causing the second leading cancer-related death worldwide. With the significant advances of high-throughput protein analysis techniques, the proteomics offered an extremely useful and versatile analytical platform for biomedical researches. In recent years, different proteomic strategies have been widely applied in the various aspects of HCC studies, ranging from screening the early diagnostic and prognostic biomarkers to in-depth investigating the underlying molecular mechanisms. In this review, we would like to systematically summarize the current applications of proteomics in hepatocellular carcinoma study, and discuss the challenges of applying proteomics in study clinical samples, as well as discuss the possible application of proteomics in precision medicine. In this review, we have systematically summarized the current applications of proteomics in hepatocellular carcinoma study, ranging from screening biomarkers to in-depth investigating the underlying molecular mechanisms. In addition, we have discussed the challenges of applying proteomics in study clinical samples, as well as the possible applications of proteomics in precision medicine. We believe that this review would help readers to be better familiar with the recent progresses of clinical proteomics, especially in the field of hepatocellular carcinoma research. Copyright © 2016 Elsevier B.V. All rights reserved.

Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins

PubMed Central

Mesquita, Rosilene Oliveira; de Almeida Soares, Eduardo; de Barros, Everaldo Gonçalves; Loureiro, Marcelo Ehlers

2012-01-01

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins. PMID:22802721

Subnanogram proteomics: impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

DOE PAGES

Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; ...

2017-09-01

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here in this study, we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap),more » leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~95% for 0.5 ng samples and by ~42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. Finally, the present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.« less

Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level.

PubMed

Bensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Murillo, Alejandro Brenes; Gartner, Anton; Kenyon, Cynthia J; Lamond, Angus I

2016-02-01

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation and Preparation of Extracellular Proteins from Lignocellulose Degrading Fungi for Comparative Proteomic Studies Using Mass Spectrometry.

PubMed

Gruninger, Robert J; Tsang, Adrian; McAllister, Tim A

2017-01-01

Fungi utilize a unique mechanism of nutrient acquisition involving extracellular digestion. To understand the biology of these microbes, it is important to identify and characterize the function of proteins that are secreted and involved in this process. Mass spectrometry-based proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins produced by an organism change in response to different conditions. Many fungi are efficient decomposers of plant cell wall, and anaerobic fungi are well recognized for their ability to digest lignocellulose. Here, we outline a protocol for the enrichment and isolation of proteins secreted by anaerobic fungi after growth on simple (glucose) and complex (straw and alfalfa hay) carbon sources. We provide detailed instruction on generating protein fragments and preparing these for proteomic analysis using reversed phase chromatography and mass spectrometry.

Laser capture microdissection to study flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena Ewa; Skarzyńska, Agnieszka; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2017-08-01

Laser Capture Microdissection (LCM) is a sample preparation microscopic method that enables isolation of an interesting cell or cells population from human, animal or plant tissue. This technique allows for obtaining pure sample from heterogeneous mixture. From isolated cells, it is possible to obtain the appropriate quality material used for genomic research in transcriptomics, proteomics and metabolomics. We used LCM method to study flower morphogenesis and specific bud's organ organization and development. The genes expression level in developing flower buds of male (B10) and female (2gg) lines were analyzed with qPCR. The expression was checked for stamen and carpel primordia obtained with LCM and for whole flower buds at successive stages of growth.

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) in orthodontic treatment: first pilot study.

PubMed

Ciavarella, Domenico; Mastrovincenzo, Mario; D'Onofrio, Valentina; Chimenti, Claudio; Parziale, Vincenzo; Barbato, Ersilia; Lo Muzio, Lorenzo

2011-11-01

SELDI-TOF-MS (Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) allows the generation of an accurate protein profile from minimal amounts of biological samples and may executes proteomic profile of saliva. The aim of this work is to compare the proteomic profile of saliva of patients in orthodontic treatment to the beginning of treatment and after three months by using the surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Saliva was collected from 14 patients, between the 11 and 17 years, to the beginning of the orthodontic treatment and after three months. Specimens were centrifuged (10 min, 13000 x g); the Q10 ProteinChips were prepared according to the manufacturer's instructions and were loaded with the supernatants. A saturated solution of sinapinic acid was used as energy-absorbing matrix. The analysis was performed in a m/z range from 2500 to 25000 Da, and the proteomic profiles were compared by a specific data analysis software. Saliva (5 mL) was collected by spitting directly into a clean 15 mL conical tube. The samples were then aliquotted and stored at -80°C until use. Profile of saliva of patients before orthodontic treatment present a number of peaks different respect profile of saliva after three months of treatment. The average intensities of peaks at m/z 3372, 5232, 4045 and 10128 were significantly higher after three months then at beginning of treatment in the same patients and among these one. The Roc Plot has demonstrated high sensitivity and specificity. Many differences were noted in salivary proteomic profile obtained using the SELDI-TOF-MS technology in patients in orthodontic treatment to beginning and after three months. These data suggest that the proteomic analysis of saliva is a promising new tool for a non-invasive study of oral mucosa and bone changes. Copyright © 2011 Società Italiana di Ortodonzia SIDO. Published by Elsevier Srl. All rights reserved.

The Proteome of Native Adult Müller Glial Cells From Murine Retina*

PubMed Central

Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca

2016-01-01

To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Müller cells in retinal pathologies — a topic still controversially discussed. PMID:26324419

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

PubMed

Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

2010-06-01

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

PubMed

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-06-23

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from genome sequences, though there are over lapped proteins. Based on the demonstrated application of data stored in the database for functional analyses, it is suggested that these data will be useful for analyses of biological mechanisms in soybean. Furthermore, coupled with recent advances in information and communication technology, the usefulness of this database would increase in the analyses of biological mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

CPTAC Biospecimen Collection Solicitation | Office of Cancer Clinical Proteomics Research

Cancer.gov

A funding opportunity in support of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) seeks to prospectively procure tumor samples, collected for proteomics investigation. The scope of work under this Statement of Work encompasses the activities needed to prospectively procure high quality, clinically annotated human tumor samples, blood and plasma, and when feasible, normal tissue from volunteer patients suffering from colon, ovarian, and breast cancer.

Comparative Testis Tissue Proteomics Using 2-Dye Versus 3-Dye DIGE Analysis.

PubMed

Holland, Ashling

2018-01-01

Comparative tissue proteomics aims to analyze alterations of the proteome in response to a stimulus. Two-dimensional difference gel electrophoresis (2D-DIGE) is a modified and advanced form of 2D gel electrophoresis. DIGE is a powerful biochemical method that compares two or three protein samples on the same analytical gel, and can be used to establish differentially expressed protein levels between healthy normal and diseased pathological tissue sample groups. Minimal DIGE labeling can be used via a 2-dye system with Cy3 and Cy5 or a 3-dye system with Cy2, Cy3, and Cy5 to fluorescently label samples with CyDye flours pre-electrophoresis. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization. This form of quantitative high-resolution proteomics facilitates the comparative analysis and evaluation of tissue protein compositions. Comparing tissue groups under different conditions is crucially important for advancing the biomedical field by characterization of cellular processes, understanding pathophysiological development and tissue biomarker discovery. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of 2-dye and 3-dye DIGE minimal labeling.

Proteome Studies of Filamentous Fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Scott E.; Panisko, Ellen A.

2011-04-20

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide breadth of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, non-gel basedmore » proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of different variations on the general method and technologies for identifying peptides in a given sample. We present a method that can serve as a “baseline” for proteomic studies of fungi.« less

Proteome studies of filamentous fungi.

PubMed

Baker, Scott E; Panisko, Ellen A

2011-01-01

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide variety of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, nongel-based proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of variations on the general methods and technologies for identifying peptides in a given sample. We present a method that can serve as a "baseline" for proteomic studies of fungi.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

PubMed

Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S

2017-05-01

The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches. © 2017 Wiley Periodicals, Inc.

Multidimensional protein identification technology (MudPIT): technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue.

PubMed

Kislinger, Thomas; Gramolini, Anthony O; MacLennan, David H; Emili, Andrew

2005-08-01

An optimized analytical expression profiling strategy based on gel-free multidimensional protein identification technology (MudPIT) is reported for the systematic investigation of biochemical (mal)-adaptations associated with healthy and diseased heart tissue. Enhanced shotgun proteomic detection coverage and improved biological inference is achieved by pre-fractionation of excised mouse cardiac muscle into subcellular components, with each organellar fraction investigated exhaustively using multiple repeat MudPIT analyses. Functional-enrichment, high-confidence identification, and relative quantification of hundreds of organelle- and tissue-specific proteins are achieved readily, including detection of low abundance transcriptional regulators, signaling factors, and proteins linked to cardiac disease. Important technical issues relating to data validation, including minimization of artifacts stemming from biased under-sampling and spurious false discovery, together with suggestions for further fine-tuning of sample preparation, are discussed. A framework for follow-up bioinformatic examination, pattern recognition, and data mining is also presented in the context of a stringent application of MudPIT for probing fundamental aspects of heart muscle physiology as well as the discovery of perturbations associated with heart failure.

High-specificity quantification method for almond-by-products, based on differential proteomic analysis.

PubMed

Zhang, Shiwei; Wang, Shifeng; Huang, Jingmin; Lai, Xintian; Du, Yegang; Liu, Xiaoqing; Li, Bifang; Feng, Ronghu; Yang, Guowu

2016-03-01

A highly specific competitive enzyme-linked immunosorbent assay (ELISA) protocol has been developed to identify and classify almond products based on differential proteomic analysis. We applied two-dimensional electrophoresis to compare the differences between almond and apricot kernels to search for almond-specific proteins. The amino acid of apricot Pru-1 was sequenced and aligned to almond Pru-1. One peptide, RQGRQQGRQQQEEGR, which exists in almond but not in apricot, was used as hapten to prepare monoclonal antibody against almond Pru-1. An optimized ELISA method was established using this antibody. The assay did not exhibit cross-reactivity with the tested apricot kernels and other edible plant seeds. The limit of detection (LOD) was 2.5-100μg/g based on different food samples. The recoveries of fortified samples at levels of twofold and eightfold LOD ranged from 82% to 96%. The coefficients of variation were less than 13.0%. Using 7M urea as extracting solution, the heat-treated protein loss ratios were 2%, 5% and 15% under pasteurization (65°C for 30min), baking (150°C for 30min) and autoclaved sterilization (120°C for 15min), respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

CPTAC Releases Largest-Ever Colorectal Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

On September 4, 2013, NCI’s Clinical Proteomics Tumor Analysis Consortium (CPTAC) publicly released proteomic data produced from colorectal tumor samples previously analyzed by The Cancer Genome Atlas (TCGA).  This is the initial release of proteomic tumor data designed to complement genomic data on the same tumors. The data is publicly available at the CPTAC data portal.

CPTAC Releases Largest-Ever Ovarian Cancer Proteome Dataset from Previously Genome Characterized Tumors | Office of Cancer Clinical Proteomics Research

Cancer.gov

National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).  This is one of the largest public datasets covering the proteome, phosphoproteome and glycoproteome with complementary deep genomic sequencing data on the same tumor.

Integrated proteomic and genomic analysis of colorectal cancer

Cancer.gov

Investigators who analyzed 95 human colorectal tumor samples have determined how gene alterations identified in previous analyses of the same samples are expressed at the protein level. The integration of proteomic and genomic data, or proteogenomics, pro

Scientific Approaches | Office of Cancer Clinical Proteomics Research

Cancer.gov

CPTAC employs two complementary scientific approaches, a "Targeting Genome to Proteome" (Targeting G2P) approach and a "Mapping Proteome to Genome" (Mapping P2G) approach, in order to address biological questions from data generated on a sample.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

PubMed

Alberio, Tiziana; Pieroni, Luisa; Ronci, Maurizio; Banfi, Cristina; Bongarzone, Italia; Bottoni, Patrizia; Brioschi, Maura; Caterino, Marianna; Chinello, Clizia; Cormio, Antonella; Cozzolino, Flora; Cunsolo, Vincenzo; Fontana, Simona; Garavaglia, Barbara; Giusti, Laura; Greco, Viviana; Lucacchini, Antonio; Maffioli, Elisa; Magni, Fulvio; Monteleone, Francesca; Monti, Maria; Monti, Valentina; Musicco, Clara; Petrosillo, Giuseppe; Porcelli, Vito; Saletti, Rosaria; Scatena, Roberto; Soggiu, Alessio; Tedeschi, Gabriella; Zilocchi, Mara; Roncada, Paola; Urbani, Andrea; Fasano, Mauro

2017-12-01

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.

PubMed

Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun

2018-04-17

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Proteomic analysis of tissue samples in translational breast cancer research.

PubMed

Gromov, Pavel; Moreira, José M A; Gromova, Irina

2014-06-01

In the last decade, many proteomic technologies have been applied, with varying success, to the study of tissue samples of breast carcinoma for protein expression profiling in order to discover protein biomarkers/signatures suitable for: characterization and subtyping of tumors; early diagnosis, and both prognosis and prediction of outcome of chemotherapy. The purpose of this review is to critically appraise what has been achieved to date using proteomic technologies and to bring forward novel strategies - based on the analysis of clinically relevant samples - that promise to accelerate the translation of basic discoveries into the daily breast cancer clinical practice. In particular, we address major issues in experimental design by reviewing the strengths and weaknesses of current proteomic strategies in the context of the analysis of human breast tissue specimens.

Current trends in quantitative proteomics - an update.

PubMed

Li, H; Han, J; Pan, J; Liu, T; Parker, C E; Borchers, C H

2017-05-01

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

PubMed Central

Licier, Rígel; Miranda, Eric; Serrano, Horacio

2016-01-01

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine. PMID:28248241

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

PubMed

Licier, Rígel; Miranda, Eric; Serrano, Horacio

2016-10-17

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

HTAPP: High-Throughput Autonomous Proteomic Pipeline

PubMed Central

Yu, Kebing; Salomon, Arthur R.

2011-01-01

Recent advances in the speed and sensitivity of mass spectrometers and in analytical methods, the exponential acceleration of computer processing speeds, and the availability of genomic databases from an array of species and protein information databases have led to a deluge of proteomic data. The development of a lab-based automated proteomic software platform for the automated collection, processing, storage, and visualization of expansive proteomic datasets is critically important. The high-throughput autonomous proteomic pipeline (HTAPP) described here is designed from the ground up to provide critically important flexibility for diverse proteomic workflows and to streamline the total analysis of a complex proteomic sample. This tool is comprised of software that controls the acquisition of mass spectral data along with automation of post-acquisition tasks such as peptide quantification, clustered MS/MS spectral database searching, statistical validation, and data exploration within a user-configurable lab-based relational database. The software design of HTAPP focuses on accommodating diverse workflows and providing missing software functionality to a wide range of proteomic researchers to accelerate the extraction of biological meaning from immense proteomic data sets. Although individual software modules in our integrated technology platform may have some similarities to existing tools, the true novelty of the approach described here is in the synergistic and flexible combination of these tools to provide an integrated and efficient analysis of proteomic samples. PMID:20336676

Improved Identification of Membrane Proteins by MALDI-TOF MS/MS Using Vacuum Sublimated Matrix Spots on an Ultraphobic Chip Surface

PubMed Central

Poetsch, Ansgar; Schlüsener, Daniela; Florizone, Christine; Eltis, Lindsay; Menzel, Christoph; Rögner, Matthias; Steinert, Kerstin; Roth, Udo

2008-01-01

Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface. In experiments using standard peptides, an up to 10-fold gain of sensitivity was found for on-chip preparations compared with classical dried-droplet preparations on a steel target. In order to assess the performance of the chips with membrane proteins, three model proteins (bacteriorhodopsin, subunit IV(a) of ATP synthase, and the cp47 subunit from photosystem II) were analyzed. To mimic realistic analysis conditions, purified proteins were separated by SDS-PAGE and digested with trypsin. The digest MALDI samples were prepared either by dried-droplet technique on steel plates using CHCA as matrix, or applied directly onto the matrix spots of the chip surface. Significantly higher signal-to-noise ratios were observed for all of the spectra resulting from on-chip preparations of different peptides. In a second series of experiments, the membrane proteome of Rhodococcus jostii RHA1 was investigated by AIEC/SDS-PAGE in combination with MALDI-TOF MS/MS. As in the first experiments, Coomassie-stained SDS-PAGE bands were digested and the two different preparation methods were compared. For preparations on the Mass·Spec·Turbo Chip, 43 of 60 proteins were identified, whereas only 30 proteins were reliably identified after classical sample preparation. Comparison of the obtained Mascot scores, which reflect the confidence level of the protein identifications, revealed that for 70% of the identified proteins, higher scores were obtained by on-chip sample preparation. Typically, this gain was a consequence of higher sequence coverage due to increased sensitivity. PMID:19137096

A proteomics assay to detect eight CBRN-relevant toxins in food.

PubMed

Gilquin, Benoit; Jaquinod, Michel; Louwagie, Mathilde; Kieffer-Jaquinod, Sylvie; Kraut, Alexandra; Ferro, Myriam; Becher, François; Brun, Virginie

2017-01-01

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of Protein Carbamylation in Urea Solution Using Ammonium Containing Buffers

PubMed Central

Sun, Shisheng; Zhou, Jian-Ying; Yang, Weiming; Zhang, Hui

2013-01-01

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N-termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium containing buffers was developed to facilitate its application in proteomic research. PMID:24161613

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes.

PubMed

Lo, Andy; Weiner, Joel H; Li, Liang

2013-09-17

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Reproducibility of combinatorial peptide ligand libraries for proteome capture evaluated by selected reaction monitoring.

PubMed

Di Girolamo, Francesco; Righetti, Pier Giorgio; Soste, Martin; Feng, Yuehan; Picotti, Paola

2013-08-26

Systems biology studies require the capability to quantify with high precision proteins spanning a broad range of abundances across multiple samples. However, the broad range of protein expression in cells often precludes the detection of low-abundance proteins. Different sample processing techniques can be applied to increase proteome coverage. Among these, combinatorial (hexa)peptide ligand libraries (CPLLs) bound to solid matrices have been used to specifically capture and detect low-abundance proteins in complex samples. To assess whether CPLL capture can be applied in systems biology studies involving the precise quantitation of proteins across a multitude of samples, we evaluated its performance across the whole range of protein abundances in Saccharomyces cerevisiae. We used selected reaction monitoring assays for a set of target proteins covering a broad abundance range to quantitatively evaluate the precision of the approach and its capability to detect low-abundance proteins. Replicated CPLL-isolates showed an average variability of ~10% in the amount of the isolated proteins. The high reproducibility of the technique was not dependent on the abundance of the protein or the amount of beads used for the capture. However, the protein-to-bead ratio affected the enrichment of specific proteins. We did not observe a normalization effect of CPLL beads on protein abundances. However, CPLLs enriched for and depleted specific sets of proteins and thus changed the abundances of proteins from a whole proteome extract. This allowed the identification of ~400 proteins otherwise undetected in an untreated sample, under the experimental conditions used. CPLL capture is thus a useful tool to increase protein identifications in proteomic experiments, but it should be coupled to the analysis of untreated samples, to maximize proteome coverage. Our data also confirms that CPLL capture is reproducible and can be confidently used in quantitative proteomic experiments. Combinatorial hexapeptide ligand libraries (CPLLs) bound to solid matrices have been proposed to specifically capture and detect low-abundance proteins in complex samples. To assess whether the CPLL capture can be confidently applied in systems biology studies involving the precise quantitation of proteins across a broad range of abundances and a multitude of samples, we evaluated its reproducibility and performance features. Using selected reaction monitoring assays for proteins covering the whole range of abundances we show that the technique is reproducible and compatible with quantitative proteomic studies. However, the protein-to-bead ratio affects the enrichment of specific proteins and CPLLs depleted specific sets of proteins from a whole proteome extract. Our results suggest that CPLL-based analyses should be coupled to the analysis of untreated samples, to maximize proteome coverage. Overall, our data confirms the applicability of CPLLs in systems biology research and guides the correct use of this technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick

2005-07-14

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there ismore » as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.« less

A comprehensive and scalable database search system for metaproteomics.

PubMed

Chatterjee, Sandip; Stupp, Gregory S; Park, Sung Kyu Robin; Ducom, Jean-Christophe; Yates, John R; Su, Andrew I; Wolan, Dennis W

2016-08-16

Mass spectrometry-based shotgun proteomics experiments rely on accurate matching of experimental spectra against a database of protein sequences. Existing computational analysis methods are limited in the size of their sequence databases, which severely restricts the proteomic sequencing depth and functional analysis of highly complex samples. The growing amount of public high-throughput sequencing data will only exacerbate this problem. We designed a broadly applicable metaproteomic analysis method (ComPIL) that addresses protein database size limitations. Our approach to overcome this significant limitation in metaproteomics was to design a scalable set of sequence databases assembled for optimal library querying speeds. ComPIL was integrated with a modified version of the search engine ProLuCID (termed "Blazmass") to permit rapid matching of experimental spectra. Proof-of-principle analysis of human HEK293 lysate with a ComPIL database derived from high-quality genomic libraries was able to detect nearly all of the same peptides as a search with a human database (~500x fewer peptides in the database), with a small reduction in sensitivity. We were also able to detect proteins from the adenovirus used to immortalize these cells. We applied our method to a set of healthy human gut microbiome proteomic samples and showed a substantial increase in the number of identified peptides and proteins compared to previous metaproteomic analyses, while retaining a high degree of protein identification accuracy and allowing for a more in-depth characterization of the functional landscape of the samples. The combination of ComPIL with Blazmass allows proteomic searches to be performed with database sizes much larger than previously possible. These large database searches can be applied to complex meta-samples with unknown composition or proteomic samples where unexpected proteins may be identified. The protein database, proteomic search engine, and the proteomic data files for the 5 microbiome samples characterized and discussed herein are open source and available for use and additional analysis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xing; Xu, Yanli; Meng, Qian

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP andmore » NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.« less

CPTAC Accelerates Precision Proteomics Biomedical Research | Office of Cancer Clinical Proteomics Research

Cancer.gov

The accurate quantitation of proteins or peptides using Mass Spectrometry (MS) is gaining prominence in the biomedical research community as an alternative method for analyte measurement. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) investigators have been at the forefront in the promotion of reproducible MS techniques, through the development and application of standardized proteomic methods for protein quantitation on biologically relevant samples.

Differential proteomic analysis of mouse macrophages exposed to adsorbate-loaded heavy fuel oil derived combustion particles using an automated sample-preparation workflow.

PubMed

Kanashova, Tamara; Popp, Oliver; Orasche, Jürgen; Karg, Erwin; Harndorf, Horst; Stengel, Benjamin; Sklorz, Martin; Streibel, Thorsten; Zimmermann, Ralf; Dittmar, Gunnar

2015-08-01

Ship diesel combustion particles are known to cause broad cytotoxic effects and thereby strongly impact human health. Particles from heavy fuel oil (HFO) operated ships are considered as particularly dangerous. However, little is known about the relevant components of the ship emission particles. In particular, it is interesting to know if the particle cores, consisting of soot and metal oxides, or the adsorbate layers, consisting of semi- and low-volatile organic compounds and salts, are more relevant. We therefore sought to relate the adsorbates and the core composition of HFO combustion particles to the early cellular responses, allowing for the development of measures that counteract their detrimental effects. Hence, the semi-volatile coating of HFO-operated ship diesel engine particles was removed by stepwise thermal stripping using different temperatures. RAW 264.7 macrophages were exposed to native and thermally stripped particles in submersed culture. Proteomic changes were monitored by two different quantitative mass spectrometry approaches, stable isotope labeling by amino acids in cell culture (SILAC) and dimethyl labeling. Our data revealed that cells reacted differently to native or stripped HFO combustion particles. Cells exposed to thermally stripped particles showed a very differential reaction with respect to the composition of the individual chemical load of the particle. The cellular reactions of the HFO particles included reaction to oxidative stress, reorganization of the cytoskeleton and changes in endocytosis. Cells exposed to the 280 °C treated particles showed an induction of RNA-related processes, a number of mitochondria-associated processes as well as DNA damage response, while the exposure to 580 °C treated HFO particles mainly induced the regulation of intracellular transport. In summary, our analysis based on a highly reproducible automated proteomic sample-preparation procedure shows a diverse cellular response, depending on the soot particle composition. In particular, it was shown that both the molecules of the adsorbate layer as well as particle cores induced strong but different effects in the exposed cells.

Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: Comparison of genomic and proteomic responses and anchoring to histopathological parameters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberemm, A., E-mail: axel.oberemm@bfr.bund.d; Ahr, H.-J.; Bannasch, P.

2009-12-01

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplasticmore » and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.« less

Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast.

PubMed

Addis, Maria Filippa; Tanca, Alessandro; Landolfo, Sara; Abbondio, Marcello; Cutzu, Raffaela; Biosa, Grazia; Pagnozzi, Daniela; Uzzau, Sergio; Mannazzu, Ilaria

2016-08-01

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

PubMed

Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

2014-12-05

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

Metabolomics and proteomics technologies to explore the herbal preparation affecting metabolic disorders using high resolution mass spectrometry.

PubMed

Zhang, Aihua; Zhou, Xiaohang; Zhao, Hongwei; Zou, Shiyu; Ma, Chung Wah; Liu, Qi; Sun, Hui; Liu, Liang; Wang, Xijun

2017-01-31

An integrative metabolomics and proteomics approach can provide novel insights in the understanding of biological systems. We have integrated proteome and metabolome data sets for a holistic view of the molecular mechanisms in disease. Using quantitative iTRAQ-LC-MS/MS proteomics coupled with UPLC-Q-TOF-HDMS based metabolomics, we determined the protein and metabolite expression changes in the kidney-yang deficiency syndrome (KYDS) rat model and further investigated the intervention effects of the Jinkui Shenqi Pill (JSP). The VIP-plot of the orthogonal PLS-DA (OPLS-DA) was used for discovering the potential biomarkers to clarify the therapeutic mechanisms of JSP in treating KYDS. The results showed that JSP can alleviate the kidney impairment induced by KYDS. Sixty potential biomarkers, including 5-l-glutamyl-taurine, phenylacetaldehyde, 4,6-dihydroxyquinoline, and xanthurenic acid etc., were definitely up- or down-regulated. The regulatory effect of JSP on the disturbed metabolic pathways was proved by the established metabonomic method. Using pathway analyses, we identified the disturbed metabolic pathways such as taurine and hypotaurine metabolism, pyrimidine metabolism, tyrosine metabolism, tryptophan metabolism, histidine metabolism, steroid hormone biosynthesis, etc. Furthermore, using iTRAQ-based quantitative proteomics analysis, seventeen differential proteins were identified and significantly altered by the JSP treatment. These proteins appear to be involved in Wnt, chemokine, PPAR, and MAPK signaling pathways, etc. Functional pathway analysis revealed that most of the proteins were found to play a key role in the regulation of metabolism pathways. Bioinformatics analysis with the IPA software found that these differentially-expressed moleculars had a strong correlation with the α-adrenergic signaling, FGF signaling, etc. Our data indicate that high-throughput metabolomics and proteomics can provide an insight on the herbal preparations affecting the metabolic disorders using high resolution mass spectrometry.

Proteomic analysis of synaptoneurosomes highlights the relevant role of local translation in the hippocampus.

PubMed

Benito, Itziar; Casañas, Juan José; Montesinos, María Luz

2018-06-19

Several proteomic analyses have been performed on synaptic fractions isolated from cortex or even total brain, resulting in preparations with a high synaptic heterogeneity and complexity. Synaptoneurosomes (SNs) are subcellular membranous elements that contain sealed pre- and post-synaptic components. They are obtained by subcellular fractionation of brain homogenates and serve as a suitable model to study many aspects of the synapse physiology. Here we report the proteomic content of SNs isolated from hippocampus of adult mice, a brain region involved in memory that presents lower synaptic heterogeneity than cortex. Interestingly, in addition to pre- and post-synaptic proteins, we found that proteins involved in RNA binding and translation were overrepresented in our preparation. These results validate the protocol we previously reported for SNs isolation, and, as reported by other authors, highlight the relevance of local synaptic translation for hippocampal physiology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

PubMed

Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

2013-05-01

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

PubMed

Stavrianakou, Maria; Perez, Ricardo; Wu, Cheng; Sachs, Matthew S; Aramayo, Rodolfo; Harlow, Mark

2017-08-14

The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

PubMed

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

Urinary proteomics in renal pathophysiology: Impact of proteinuria.

PubMed

Sancho-Martínez, Sandra M; Prieto-García, Laura; Blanco-Gozalo, Víctor; Fontecha-Barriuso, Miguel; López-Novoa, José M; López-Hernández, Francisco J

2015-06-01

Urinary differential proteomics is used to study renal pathophysiological mechanisms, find novel markers of biological processes and renal diseases, and stratify patients according to proteomic profiles. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results. Urine samples for differential proteomic studies are usually normalized by protein content, regardless of its pathophysiological characteristics. In the field of nephrology, this approach translates into the comparison of a different fraction of the total daily urine output between proteinuric and nonproteinuric samples. Accordingly, alterations in the level of specific proteins found by this method reflect the relative presence of individual proteins in the urine; but they do not necessarily show alterations in their daily excretion, which is a key parameter for the understanding of the pathophysiological meaning of urinary components. For renal pathophysiology studies and clinical biomarker identification or determination, an alternative proteomic concept providing complementary information is based on sample normalization by daily urine output, which directly informs on changes in the daily excretion of individual proteins. This is clinically important because daily excretion (rather than absolute or relative concentration) is the only self-normalized way to evaluate the real meaning of urinary parameters, which is also independent of urine concentration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Recent advances in metabonomics].

PubMed

Xu, Guo-Wang; Lu, Xin; Yang, Sheng-Li

2007-12-01

Metabonomics (or metabolomics) aims at the comprehensive and quantitative analysis of the wide arrays of metabolites in biological samples. Metabonomics has been labeled as one of the new" -omics" joining genomics, transcriptomics, and proteomics as a science employed toward the understanding of global systems biology. It has been widely applied in many research areas including drug toxicology, biomarker discovery, functional genomics, and molecular pathology etc. The comprehensive analysis of the metabonome is particularly challenging due to the diverse chemical natures of metabolites. Metabonomics investigations require special approaches for sample preparation, data-rich analytical chemical measurements, and information mining. The outputs from a metabonomics study allow sample classification, biomarker discovery, and interpretation of the reasons for classification information. This review focuses on the currently new advances in various technical platforms of metabonomics and its applications in drug discovery and development, disease biomarker identification, plant and microbe related fields.

Towards proteomic analysis of milk proteins in historical building materials

NASA Astrophysics Data System (ADS)

Kuckova, S.; Crhova, M.; Vankova, L.; Hnizda, A.; Hynek, R.; Kodicek, M.

2009-07-01

The addition of proteinaceous binders to mortars and plasters has a long tradition. The protein additions were identified in many sacral and secular historical buildings. For this method of peptide mass mapping, three model mortar samples with protein additives were prepared. These samples were analysed fresh (1-2 weeks old) and after 9 months of natural ageing. The optimal duration of tryptic cleavage (2 h) and the lowest amount of material needed for relevant analysis of fresh and weathered samples were found; the sufficient amounts of weathered and fresh mortars were set to 0.05 and 0.005 g. The list of main tryptic peptides coming from milk additives (bovine milk, curd, and whey), their relative intensities and theoretical amino acid sequences assignment is presented. Several sequences have been "de novo" confirmed by mass spectrometry.

[Laser microdissection for biology and medicine].

PubMed

Podgornyĭ, O V; Lazarev, V N; Govorun, V M

2012-01-01

For routine extraction of DNA, RNA, proteins and metabolites, small tissue pieces are placed into lysing solution. These tissue pieces in general contain different cell types. For this reason, lysate contains components of different cell types, which complicates the interpretation of molecular analysis results. The laser microdissection allows overcoming this trouble. The laser microdissection is a method to procure tissue samples contained defined cell subpopulations, individual cells and even subsellular components under direct microscopic visualization. Collected samples can be undergone to different downstream molecular assays: DNA analysis, RNA transcript profiling, cDNA library generation and gene expression analysis, proteomic analysis and metabolite profiling. The laser microdissection has wide applications in oncology (research and routine), cellular and molecular biology, biochemistry and forensics. This paper reviews the principles of different laser microdissection instruments, examples of laser microdissection application and problems of sample preparation for laser microdissection.

Modulating the protein content of complex proteomes using acetonitrile.

PubMed

Prates, João; Martins, Gonçalo; López-Fernández, Hugo; Lodeiro, Carlos; Capelo, J L; Santos, Hugo M

2018-05-15

In this work we present acetonitrile as a tool to modulate the dynamic range of the proteome of complex samples. Different concentrations of acetonitrile ranging from 15% v/v to 65% v/v were used to modulate the protein content of serum samples from healthy people and patients with lymphoma and myeloma. We show that the proteome above 70 kDa is pelleted as a function of the concentration of acetonitrile and that profiling with PCA or Clustering is only possible using the supernatants obtained for concentrations of acetonitrile higher than 45% v/v or the pellets for concentrations of acetonitrile of 35% and 45%. The differentiation and classification of the three groups of sera samples (healthy, lymphoma and myeloma) were possible using acetonitrile at 55% v/v concentration. This work opens new avenues for the application of acetonitrile as a cost-effective tool in proteomics applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of individual mouse cerebrospinal fluid proteomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles

2014-03-20

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% falsemore » discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Denef, Vincent; Shah, Manesh B; Verberkmoes, Nathan C

The recent surge in microbial genomic sequencing, combined with the development of high-throughput liquid chromatography-mass-spectrometry-based (LC/LC-MS/MS) proteomics, has raised the question of the extent to which genomic information of one strain or environmental sample can be used to profile proteomes of related strains or samples. Even with decreasing sequencing costs, it remains impractical to obtain genomic sequence for every strain or sample analyzed. Here, we evaluate how shotgun proteomics is affected by amino acid divergence between the sample and the genomic database using a probability-based model and a random mutation simulation model constrained by experimental data. To assess the effectsmore » of nonrandom distribution of mutations, we also evaluated identification levels using in silico peptide data from sequenced isolates with average amino acid identities (AAI) varying between 76 and 98%. We compared the predictions to experimental protein identification levels for a sample that was evaluated using a database that included genomic information for the dominant organism and for a closely related variant (95% AAI). The range of models set the boundaries at which half of the proteins in a proteomic experiment can be identified to be 77-92% AAI between orthologs in the sample and database. Consistent with this prediction, experimental data indicated loss of half the identifiable proteins at 90% AAI. Additional analysis indicated a 6.4% reduction of the initial protein coverage per 1% amino acid divergence and total identification loss at 86% AAI. Consequently, shotgun proteomics is capable of cross-strain identifications but avoids most crossspecies false positives.« less

P2P proteomics -- data sharing for enhanced protein identification

PubMed Central

2012-01-01

Background In order to tackle the important and challenging problem in proteomics of identifying known and new protein sequences using high-throughput methods, we propose a data-sharing platform that uses fully distributed P2P technologies to share specifications of peer-interaction protocols and service components. By using such a platform, information to be searched is no longer centralised in a few repositories but gathered from experiments in peer proteomics laboratories, which can subsequently be searched by fellow researchers. Methods The system distributively runs a data-sharing protocol specified in the Lightweight Communication Calculus underlying the system through which researchers interact via message passing. For this, researchers interact with the system through particular components that link to database querying systems based on BLAST and/or OMSSA and GUI-based visualisation environments. We have tested the proposed platform with data drawn from preexisting MS/MS data reservoirs from the 2006 ABRF (Association of Biomolecular Resource Facilities) test sample, which was extensively tested during the ABRF Proteomics Standards Research Group 2006 worldwide survey. In particular we have taken the data available from a subset of proteomics laboratories of Spain's National Institute for Proteomics, ProteoRed, a network for the coordination, integration and development of the Spanish proteomics facilities. Results and Discussion We performed queries against nine databases including seven ProteoRed proteomics laboratories, the NCBI Swiss-Prot database and the local database of the CSIC/UAB Proteomics Laboratory. A detailed analysis of the results indicated the presence of a protein that was supported by other NCBI matches and highly scored matches in several proteomics labs. The analysis clearly indicated that the protein was a relatively high concentrated contaminant that could be present in the ABRF sample. This fact is evident from the information that could be derived from the proposed P2P proteomics system, however it is not straightforward to arrive to the same conclusion by conventional means as it is difficult to discard organic contamination of samples. The actual presence of this contaminant was only stated after the ABRF study of all the identifications reported by the laboratories. PMID:22293032

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

PubMed

Prieto, DaRue A; Chan, King C; Johann, Donald J; Ye, Xiaoying; Whitely, Gordon; Blonder, Josip

2017-01-01

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

A Protocol for the Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Studies.

PubMed

Sparrow, Rosemary L; Simpson, Richard J; Greening, David W

2017-01-01

Cryoprecipitate is a concentrate of high-molecular-weight plasma proteins that precipitate when frozen plasma is slowly thawed at 1-6 °C. The concentrate contains factor VIII (antihemophilic factor), von Willebrand factor (vWF), fibrinogen, factor XIII, fibronectin, and small amounts of other plasma proteins. Clinical grade preparations of cryoprecipitate are mainly used to treat fibrinogen deficiency caused by acute bleeding or functional abnormalities of the fibrinogen protein. In the past, cryoprecipitate was used to treat von Willebrand disease and hemophilia A (factor VIII deficiency), but the availability of more highly purified coagulation factor concentrates or recombinant protein preparations has superseded the use of cryoprecipitate for these coagulopathies. Cryo-depleted plasma ("cryosupernatant") is the plasma supernatant remaining following removal of the cryoprecipitate from frozen-thawed plasma. It contains all the other plasma proteins and clotting factors present in plasma that remain soluble during cold-temperature thawing of the plasma. This protocol describes the clinical-scale preparation of cryoprecipitate and cryo-depleted plasma for proteomic studies.

Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-linked Glycans

PubMed Central

Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.

2013-01-01

Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204

Xylem sap proteomics.

PubMed

de Bernonville, Thomas Dugé; Albenne, Cécile; Arlat, Matthieu; Hoffmann, Laurent; Lauber, Emmanuelle; Jamet, Elisabeth

2014-01-01

Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Feasibility of investigating differential proteomic expression in depression: implications for biomarker development in mood disorders

PubMed Central

Frye, M A; Nassan, M; Jenkins, G D; Kung, S; Veldic, M; Palmer, B A; Feeder, S E; Tye, S J; Choi, D S; Biernacka, J M

2015-01-01

The objective of this study was to determine whether proteomic profiling in serum samples can be utilized in identifying and differentiating mood disorders. A consecutive sample of patients with a confirmed diagnosis of unipolar (UP n=52) or bipolar depression (BP-I n=46, BP-II n=49) and controls (n=141) were recruited. A 7.5-ml blood sample was drawn for proteomic multiplex profiling of 320 proteins utilizing the Myriad RBM Discovery Multi-Analyte Profiling platform. After correcting for multiple testing and adjusting for covariates, growth differentiation factor 15 (GDF-15), hemopexin (HPX), hepsin (HPN), matrix metalloproteinase-7 (MMP-7), retinol-binding protein 4 (RBP-4) and transthyretin (TTR) all showed statistically significant differences among groups. In a series of three post hoc analyses correcting for multiple testing, MMP-7 was significantly different in mood disorder (BP-I+BP-II+UP) vs controls, MMP-7, GDF-15, HPN were significantly different in bipolar cases (BP-I+BP-II) vs controls, and GDF-15, HPX, HPN, RBP-4 and TTR proteins were all significantly different in BP-I vs controls. Good diagnostic accuracy (ROC-AUC⩾0.8) was obtained most notably for GDF-15, RBP-4 and TTR when comparing BP-I vs controls. While based on a small sample not adjusted for medication state, this discovery sample with a conservative method of correction suggests feasibility in using proteomic panels to assist in identifying and distinguishing mood disorders, in particular bipolar I disorder. Replication studies for confirmation, consideration of state vs trait serial assays to delineate proteomic expression of bipolar depression vs previous mania, and utility studies to assess proteomic expression profiling as an advanced decision making tool or companion diagnostic are encouraged. PMID:26645624

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

PubMed

Zhang, Lina; de Waard, Marita; Verheijen, Hester; Boeren, Sjef; Hageman, Jos A; van Hooijdonk, Toon; Vervoort, Jacques; van Goudoever, Johannes B; Hettinga, Kasper

2016-09-16

To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the quantitative level than qualitative level. The correlation between the changes of milk proteins and the gradual maturation of the gastrointestinal tract and immune system in infants, contributes to a better understanding of the biological functions of human milk proteins for the growth and development of infants. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

PubMed

Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

2005-07-01

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

mzResults: An Interactive Viewer for Interrogation and Distribution of Proteomics Results*

PubMed Central

Webber, James T.; Askenazi, Manor; Marto, Jarrod A.

2011-01-01

The growing use of mass spectrometry in the context of biomedical research has been accompanied by an increased demand for distribution of results in a format that facilitates rapid and efficient validation of claims by reviewers and other interested parties. However, the continued evolution of mass spectrometry hardware, sample preparation methods, and peptide identification algorithms complicates standardization and creates hurdles related to compliance with journal submission requirements. Moreover, the recently announced Philadelphia Guidelines (1, 2) suggest that authors provide native mass spectrometry data files in support of their peer-reviewed research articles. These trends highlight the need for data viewers and other tools that work independently of manufacturers' proprietary data systems and seamlessly connect proteomics results with original data files to support user-driven data validation and review. Based upon our recently described API1-based framework for mass spectrometry data analysis (3, 4), we created an interactive viewer (mzResults) that is built on established database standards and enables efficient distribution and interrogation of results associated with proteomics experiments, while also providing a convenient mechanism for authors to comply with data submission standards as described in the Philadelphia Guidelines. In addition, the architecture of mzResults supports in-depth queries of the native mass spectrometry files through our multiplierz software environment. We use phosphoproteomics data to illustrate the features and capabilities of mzResults. PMID:21266631

Proteomics of blood-based therapeutics: a promising tool for quality assurance in transfusion medicine.

PubMed

Thiele, Thomas; Steil, Leif; Völker, Uwe; Greinacher, Andreas

2007-01-01

Blood-based therapeutics are cellular or plasma components derived from human blood. Their production requires appropriate selection and treatment of the donor and processing of cells or plasma proteins. In contrast to clearly defined, chemically synthesized drugs, blood-derived therapeutics are highly complex mixtures of plasma proteins or even more complex cells. Pathogen transmission by the product as well as changes in the integrity of blood constituents resulting in loss of function or immune modulation are currently important issues in transfusion medicine. Protein modifications can occur during various steps of the production process, such as acquisition, enrichment of separate components (e.g. coagulation factors, cell populations), virus inactivation, conservation, and storage. Contemporary proteomic strategies allow a comprehensive assessment of protein modifications with high coverage, offer capabilities for qualitative and even quantitative analysis, and for high-throughput protein identification. Traditionally, proteomics approaches predominantly relied on two-dimensional gel electrophoresis (2-DE). Even if 2-DE is still state of the art, it has inherent limitations that are mainly based on the physicochemical properties of the proteins analyzed; for example, proteins with extremes in molecular mass and hydrophobicity (most membrane proteins) are difficult to assess by 2-DE. These limitations have fostered the development of mass spectrometry centered on non-gel-based separation approaches, which have proven to be highly successful and are thus complementing and even partially replacing 2-DE-based approaches. Although blood constituents have been extensively analyzed by proteomics, this technology has not been widely applied to assess or even improve blood-derived therapeutics, or to monitor the production processes. As proteomic technologies have the capacity to provide comprehensive information about changes occurring during processing and storage of blood products, proteomics can potentially guide improvement of pathogen inactivation procedures and engineering of stem cells, and may also allow a better understanding of factors influencing the immunogenicity of blood-derived therapeutics. An important development in proteomics is the reduction of inter-assay variability. This now allows the screening of samples taken from the same product over time or before and after processing. Optimized preparation procedures and storage conditions will reduce the risk of protein alterations, which in turn may contribute to better recovery, reduced exposure to allogeneic proteins, and increased transfusion safety.

A DATABASE FOR TRACKING TOXICOGENOMIC SAMPLES AND PROCEDURES WITH GENOMIC, PROTEOMIC AND METABONOMIC COMPONENTS

EPA Science Inventory

A Database for Tracking Toxicogenomic Samples and Procedures with Genomic, Proteomic and Metabonomic Components
Wenjun Bao1, Jennifer Fostel2, Michael D. Waters2, B. Alex Merrick2, Drew Ekman3, Mitchell Kostich4, Judith Schmid1, David Dix1
Office of Research and Developmen...

Proteome Characterization Centers - TCGA

Cancer.gov

The centers, a component of NCI’s Clinical Proteomic Tumor Analysis Consortium, will analyze a subset of TCGA samples to define proteins translated from cancer genomes and their related biological processes.

Next-Generation Proteomics and Its Application to Clinical Breast Cancer Research.

PubMed

Mardamshina, Mariya; Geiger, Tamar

2017-10-01

Proteomics technology aims to map the protein landscapes of biological samples, and it can be applied to a variety of samples, including cells, tissues, and body fluids. Because the proteins are the main functional molecules in the cells, their levels reflect much more accurately the cellular phenotype and the regulatory processes within them than gene levels, mutations, and even mRNA levels. With the advancement in the technology, it is possible now to obtain comprehensive views of the biological systems and to study large patient cohorts in a streamlined manner. In this review we discuss the technological advancements in mass spectrometry-based proteomics, which allow analysis of breast cancer tissue samples, leading to the first large-scale breast cancer proteomics studies. Furthermore, we discuss the technological developments in blood-based biomarker discovery, which provide the basis for future development of assays for routine clinical use. Although these are only the first steps in implementation of proteomics into the clinic, extensive collaborative work between these worlds will undoubtedly lead to major discoveries and advances in clinical practice. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

PubMed Central

2012-01-01

Background Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis. Methods Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. Results A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein). Conclusions A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC. PMID:22726388

Proteomic biomarkers for ovarian cancer risk in women with polycystic ovary syndrome: a systematic review and biomarker database integration.

PubMed

Galazis, Nicolas; Olaleye, Olalekan; Haoula, Zeina; Layfield, Robert; Atiomo, William

2012-12-01

To review and identify possible biomarkers for ovarian cancer (OC) in women with polycystic ovary syndrome (PCOS). Systematic literature searches of MEDLINE, EMBASE, and Cochrane using the search terms "proteomics," "proteomic," and "ovarian cancer" or "ovarian carcinoma." Proteomic biomarkers for OC were then integrated with an updated previously published database of all proteomic biomarkers identified to date in patients with PCOS. Academic department of obstetrics and gynecology in the United Kingdom. A total of 180 women identified in the six studies. Tissue samples from women with OC vs. tissue samples from women without OC. Proteomic biomarkers, proteomic technique used, and methodologic quality score. A panel of six biomarkers was overexpressed both in women with OC and in women with PCOS. These biomarkers include calreticulin, fibrinogen-γ, superoxide dismutase, vimentin, malate dehydrogenase, and lamin B2. These biomarkers could help improve our understanding of the links between PCOS and OC and could potentially be used to identify subgroups of women with PCOS at increased risk of OC. More studies are required to further evaluate the role these biomarkers play in women with PCOS and OC. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Advanced proteomic liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Fang; Smith, Richard D.; Shen, Yufeng

2012-10-26

Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

PubMed

Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

2011-12-01

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

Proteomic analysis of heat treated bitter gourd (Momordica charantia L. var. Hong Kong Green) using 2D-DIGE.

PubMed

Ng, Zhi Xiang; Chua, Kek Heng; Kuppusamy, Umah Rani

2014-04-01

This study aimed to investigate the changes in the proteome of bitter gourd prior to and after subjecting to boiling and microwaving. A comparative analysis of the proteome profiles of raw and thermally treated bitter gourds was performed using 2D-DIGE. The protein content and number of protein spots in raw sample was higher when compared to the cooked samples. Qualitative analysis revealed that 103 (boiled sample) and 110 (microwaved sample) protein spots were up regulated whereas 120 (boiled sample) and 107 (microwaved sample) protein spots were down regulated. Ten protein spots with the highest significant fold change in the cooked samples were involved in carbohydrate/energy metabolisms and stress responses. Small heat shock proteins, superoxide dismutase, quinone oxidoreductase, UDP-glucose pyrophosphorylase and phosphoglycerate kinase play a role in heat-stress-mediated protection of bitter gourd. This study suggests that appropriate heat treatment (cooking methods) can lead to induction of selected proteins in bitter gourd. Copyright © 2013 Elsevier Ltd. All rights reserved.

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

PubMed

Leszczynski, Dariusz; de Pomerai, David; Koczan, Dirk; Stoll, Dieter; Franke, Helmut; Albar, Juan Pablo

2012-08-01

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens.

PubMed

Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

2015-01-01

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.

Inhibition of protein carbamylation in urea solution using ammonium-containing buffers.

PubMed

Sun, Shisheng; Zhou, Jian-Ying; Yang, Weiming; Zhang, Hui

2014-02-01

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research. Copyright © 2013 Elsevier Inc. All rights reserved.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

PubMed

Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE PAGES

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui; ...

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

PubMed

Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

PubMed

Lledías, Fernando; Hernández, Felipe; Rivas, Viridiana; García-Mendoza, Abisaí; Cassab, Gladys I; Nieto-Sotelo, Jorge

2017-08-01

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

Inactivation of West Nile Virus in Serum with Heat, Ionic Detergent, and Reducing Agent for Proteomic Applications (Open Access Publisher’s Version)

DTIC Science & Technology

2017-05-19

LightCycler® 96 desktop software. Positive and negative samples were identified using the “ Qualitative Detection” analysis function using the default...Institute of Infectious Diseases, Fort Detrick, MD 21702, United States A R T I C L E I N F O Keywords: West Nile virus Virus inactivation Sample buffer... samples using a commercially available SDS- PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification

Advanced proteomic liquid chromatography

PubMed Central

Xie, Fang; Smith, Richard D.; Shen, Yufeng

2012-01-01

Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput. PMID:22840822

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

PubMed

Welker, F

2018-02-20

The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identifications at increased evolutionary distances due to a larger number of protein sequence differences between the database sequence and the analyzed organism. Error-tolerant proteomic search algorithms should theoretically overcome this problem at both the peptide and protein level; however, this has not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against sequence databases at increasing evolutionary distances: the human (0 Ma), chimpanzee (6-8 Ma) and orangutan (16-17 Ma) reference proteomes, respectively. Incorrectly suggested amino acid substitutions are absent when employing adequate filtering criteria for mutable Peptide Spectrum Matches (PSMs), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations between the target and database sequences are the main factors influencing mutable PSM identification. The error-tolerant results suggest that the cross-species proteomics problem is not overcome at increasing evolutionary distances, even at the protein level. Peptide and protein loss has the potential to significantly impact divergence dating and proteome comparisons when using ancient samples as there is a bias towards the identification of conserved sequences and proteins. Effects are minimized between moderately divergent proteomes, as indicated by almost complete recovery of informative positions in the search against the chimpanzee proteome (≈90%, 6-8 Ma). This provides a bioinformatic background to future phylogenetic and proteomic analysis of ancient hominin proteomes, including the future description of novel hominin amino acid sequences, but also has negative implications for the study of fast-evolving proteins in hominins, non-hominin animals, and ancient bacterial proteins in evolutionary contexts.

Vascular Sap Proteomics: Providing Insight into Long-Distance Signaling during Stress

PubMed Central

Carella, Philip; Wilson, Daniel C.; Kempthorne, Christine J.; Cameron, Robin K.

2016-01-01

The plant vascular system, composed of the xylem and phloem, is important for the transport of water, mineral nutrients, and photosynthate throughout the plant body. The vasculature is also the primary means by which developmental and stress signals move from one organ to another. Due to practical and technological limitations, proteomics analysis of xylem and phloem sap has been understudied in comparison to accessible sample types such as leaves and roots. However, recent advances in sample collection techniques and mass spectrometry technology are making it possible to comprehensively analyze vascular sap proteomes. In this mini-review, we discuss the emerging field of vascular sap proteomics, with a focus on recent comparative studies to identify vascular proteins that may play roles in long-distance signaling and other processes during stress responses in plants. PMID:27242852

Evaluation of the salivary proteome as a surrogate tissue for systems biology approaches to understanding appetite.

PubMed

Harden, Charlotte J; Perez-Carrion, Kristine; Babakordi, Zara; Plummer, Sue F; Hepburn, Natalie; Barker, Margo E; Wright, Phillip C; Evans, Caroline A; Corfe, Bernard M

2012-06-06

Current measurement of appetite depends upon tools that are either subjective (visual analogue scales), or invasive (blood). Saliva is increasingly recognised as a valuable resource for biomarker analysis. Proteomics workflows may provide alternative means for the assessment of appetitive response. The study aimed to assess the potential value of the salivary proteome to detect novel biomarkers of appetite using an iTRAQ-based workflow. Diurnal variation of salivary protein concentrations was assessed. A randomised, controlled, crossover study examined the effects on the salivary proteome of isocaloric doses of various long chain fatty acid (LCFA) oil emulsions compared to no treatment (NT). Fasted males provided saliva samples before and following NT or dosing with LCFA emulsions. The oil component of the DHA emulsion contained predominantly docosahexaenoic acid and the oil component of OA contained predominantly oleic acid. Several proteins were present in significantly (p<0.05) different quantities in saliva samples taken following treatments compared to fasting samples. DHA caused alterations in thioredoxin and serpin B4 relative to OA and NT. A further study evaluated energy intake (EI) in response to LCFA in conjunction with subjective appetite scoring. DHA was associated with significantly lower EI relative to NT and OA (p=0.039). The collective data suggest investigation of salivary proteome may be of value in appetitive response. This article is part of a Special Issue entitled: Proteomics: The clinical link. Copyright © 2011 Elsevier B.V. All rights reserved.

A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis.

PubMed

Zhang, Xu; Liu, Qun; Zhou, Wei; Li, Ping; Alolga, Raphael N; Qi, Lian-Wen; Yin, Xiaojian

2018-06-15

Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation. C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis. Copyright © 2018. Published by Elsevier B.V.

Mining the human urine proteome for monitoring renal transplant injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sigdel, Tara K.; Gao, Yuqian; He, Jintang

The human urinary proteome reflects systemic and inherent renal injury perturbations and can be analyzed to harness specific biomarkers for different kidney transplant injury states. 396 unique urine samples were collected contemporaneously with an allograft biopsy from 396 unique kidney transplant recipients. Centralized, blinded histology on the graft was used to classify matched urine samples into categories of acute rejection (AR), chronic allograft nephropathy (CAN), BK virus nephritis (BKVN), and stable graft (STA). Liquid chromatography–mass spectrometry (LC-MS) based proteomics using iTRAQ based discovery (n=108) and global label-free LC-MS analyses of individual samples (n=137) for quantitative proteome assessment were used inmore » the discovery step. Selected reaction monitoring (SRM) was applied to identify and validate minimal urine protein/peptide biomarkers to accurately segregate organ injury causation and pathology on unique urine samples (n=151). A total of 958 proteins were initially quantified by iTRAQ, 87% of which were also identified among 1574 urine proteins detected in LC-MS validation. 103 urine proteins were significantly (p<0.05) perturbed in injury and enriched for humoral immunity, complement activation, and lymphocyte trafficking. A set of 131 peptides corresponding to 78 proteins were assessed by SRM for their significance in an independent sample cohort. A minimal set of 35 peptides mapping to 33 proteins, were modeled to segregate different injury groups (AUC =93% for AR, 99% for CAN, 83% for BKVN). Urinary proteome discovery and targeted validation identified urine protein fingerprints for non-invasive differentiation of kidney transplant injuries, thus opening the door for personalized immune risk assessment and therapy.« less

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

PubMed

Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

2015-11-06

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy.

PubMed

Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

2017-08-15

Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Salivary biomarker development using genomic, proteomic and metabolomic approaches

PubMed Central

2012-01-01

The use of saliva as a diagnostic sample provides a non-invasive, cost-efficient method of sample collection for disease screening without the need for highly trained professionals. Saliva collection is far more practical and safe compared with invasive methods of sample collection, because of the infection risk from contaminated needles during, for example, blood sampling. Furthermore, the use of saliva could increase the availability of accurate diagnostics for remote and impoverished regions. However, the development of salivary diagnostics has required technical innovation to allow stabilization and detection of analytes in the complex molecular mixture that is saliva. The recent development of cost-effective room temperature analyte stabilization methods, nucleic acid pre-amplification techniques and direct saliva transcriptomic analysis have allowed accurate detection and quantification of transcripts found in saliva. Novel protein stabilization methods have also facilitated improved proteomic analyses. Although candidate biomarkers have been discovered using epigenetic, transcriptomic, proteomic and metabolomic approaches, transcriptomic analyses have so far achieved the most progress in terms of sensitivity and specificity, and progress towards clinical implementation. Here, we review recent developments in salivary diagnostics that have been accomplished using genomic, transcriptomic, proteomic and metabolomic approaches. PMID:23114182

The Escherichia coli Proteome: Past, Present, and Future Prospects†

PubMed Central

Han, Mee-Jung; Lee, Sang Yup

2006-01-01

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

QC-ART: A tool for real-time quality control assessment of mass spectrometry-based proteomics data.

PubMed

Stanfill, Bryan A; Nakayasu, Ernesto S; Bramer, Lisa M; Thompson, Allison M; Ansong, Charles K; Clauss, Therese; Gritsenko, Marina A; Monroe, Matthew E; Moore, Ronald J; Orton, Daniel J; Piehowski, Paul D; Schepmoes, Athena A; Smith, Richard D; Webb-Robertson, Bobbie-Jo; Metz, Thomas O

2018-04-17

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those due to collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected.  In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality due to the need for instrument cleaning and/or re-calibration.  To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired in order to dynamically flag potential issues with instrument performance or sample quality.  QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis.  We demonstrate the utility and performance of QC-ART in identifying deviations in data quality due to both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A Proteomic Analysis of Eccrine Sweat: Implications for the Discovery of Schizophrenia Biomarker Proteins

PubMed Central

Raiszadeh, Michelle M.; Ross, Mark M.; Russo, Paul S.; Schaepper, Mary Ann H.; Zhou, Weidong; Deng, Jianghong; Ng, Daniel; Dickson, April; Dickson, Cindy; Strom, Monica; Osorio, Carolina; Soeprono, Thomas; Wulfkuhle, Julia D.; Kabbani, Nadine; Petricoin, Emanuel F.; Liotta, Lance A.; Kirsch, Wolff M.

2012-01-01

Liquid chromatography tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate, and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately two-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers. PMID:22256890

Advances in microscale separations towards nanoproteomics applications

DOE PAGES

Yi, Lian; Piehowski, Paul D.; Shi, Tujin; ...

2017-07-21

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. But, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1 μg total proteins (e.g., cellular heterogeneity in tissue pathologies). We review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, andmore » their contributions towards nanoproteomics applications.« less

Advances in microscale separations towards nanoproteomics applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Lian; Piehowski, Paul D.; Shi, Tujin

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. But, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1 μg total proteins (e.g., cellular heterogeneity in tissue pathologies). We review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, andmore » their contributions towards nanoproteomics applications.« less

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Chen; Hettich, Robert L.

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

DOE PAGES

Qian, Chen; Hettich, Robert L.

2017-05-24

The microbial composition and their activities in soil environments play a critical role in organic matter transformation and nutrient cycling, perhaps most specifically with respect to impact on plant growth but also more broadly to global impact on carbon and nitrogen-cycling. Liquid chromatography coupled to high performance mass spectrometry provides a powerful approach to characterize soil microbiomes; however, the limited microbial biomass and the presence of abundant interferences in soil samples present major challenges to soil proteome extraction and subsequent MS measurement. To address some of the major issues, we have designed and optimized an experimental method to enhance microbialmore » proteome extraction concomitant with minimizing the soil-borne humic substances co-extraction from soils. Among the range of interferences, humic substances are often the worst in terms of adversely impacting proteome extraction and mass spectrometry measurement. Our approach employs an in-situ detergent-based microbial lysis / TCA precipitation coupled with an additional acidification precipitation step at the peptide level which efficiently removes humic acids. By combing filtration and pH adjustment of the final peptide solution, the remaining humic acids can be differentially precipitated and removed with a membrane filter, thereby leaving much cleaner proteolytic peptide samples for MS measurement. As a result, this modified method is a reliable and straight-forward protein extraction method that efficiently removes soil-borne humic substances without inducing proteome sample loss or reducing or biasing protein identification in mass spectrometry.« less

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.

PubMed

Milles, J; Krämer, J; Prange, A

2007-12-01

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

Sequential protein extraction as an efficient method for improved proteome coverage in larvae of Atlantic salmon (Salmo salar).

PubMed

Nuez-Ortín, Waldo G; Carter, Chris G; Nichols, Peter D; Wilson, Richard

2016-07-01

Understanding diet- and environmentally induced physiological changes in fish larvae is a major goal for the aquaculture industry. Proteomic analysis of whole fish larvae comprising multiple tissues offers considerable potential but is challenging due to the very large dynamic range of protein abundance. To extend the coverage of the larval phase of the Atlantic salmon (Salmo salar) proteome, we applied a two-step sequential extraction (SE) method, based on differential protein solubility, using a nondenaturing buffer containing 150 mM NaCl followed by a denaturing buffer containing 7 M urea and 2 M thiourea. Extracts prepared using SE and one-step direct extraction were characterized via label-free shotgun proteomics using nanoLC-MS/MS (LTQ-Orbitrap). SE partitioned the proteins into two fractions of approximately equal amounts, but with very distinct protein composition, leading to identification of ∼40% more proteins than direct extraction. This fractionation strategy enabled the most detailed characterization of the salmon larval proteome to date and provides a platform for greater understanding of physiological changes in whole fish larvae. The MS data are available via the ProteomeXchange Consortium PRIDE partner repository, dataset PXD003366. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomic biomarker discovery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Zhou, Jianying; Gritsenko, Marina A.

2012-02-01

Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up tomore » 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.« less

Mass spectrometry-assisted gel-based proteomics in cancer biomarker discovery: approaches and application

PubMed Central

Huang, Rongrong; Chen, Zhongsi; He, Lei; He, Nongyue; Xi, Zhijiang; Li, Zhiyang; Deng, Yan; Zeng, Xin

2017-01-01

There is a critical need for the discovery of novel biomarkers for early detection and targeted therapy of cancer, a major cause of deaths worldwide. In this respect, proteomic technologies, such as mass spectrometry (MS), enable the identification of pathologically significant proteins in various types of samples. MS is capable of high-throughput profiling of complex biological samples including blood, tissues, urine, milk, and cells. MS-assisted proteomics has contributed to the development of cancer biomarkers that may form the foundation for new clinical tests. It can also aid in elucidating the molecular mechanisms underlying cancer. In this review, we discuss MS principles and instrumentation as well as approaches in MS-based proteomics, which have been employed in the development of potential biomarkers. Furthermore, the challenges in validation of MS biomarkers for their use in clinical practice are also reviewed. PMID:28912895

Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.

PubMed

Li, Ming; Gray, William; Zhang, Haixia; Chung, Christine H; Billheimer, Dean; Yarbrough, Wendell G; Liebler, Daniel C; Shyr, Yu; Slebos, Robbert J C

2010-08-06

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher's Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples.

Comparative Shotgun Proteomics Using Spectral Count Data and Quasi-Likelihood Modeling

PubMed Central

2010-01-01

Shotgun proteomics provides the most powerful analytical platform for global inventory of complex proteomes using liquid chromatography−tandem mass spectrometry (LC−MS/MS) and allows a global analysis of protein changes. Nevertheless, sampling of complex proteomes by current shotgun proteomics platforms is incomplete, and this contributes to variability in assessment of peptide and protein inventories by spectral counting approaches. Thus, shotgun proteomics data pose challenges in comparing proteomes from different biological states. We developed an analysis strategy using quasi-likelihood Generalized Linear Modeling (GLM), included in a graphical interface software package (QuasiTel) that reads standard output from protein assemblies created by IDPicker, an HTML-based user interface to query shotgun proteomic data sets. This approach was compared to four other statistical analysis strategies: Student t test, Wilcoxon rank test, Fisher’s Exact test, and Poisson-based GLM. We analyzed the performance of these tests to identify differences in protein levels based on spectral counts in a shotgun data set in which equimolar amounts of 48 human proteins were spiked at different levels into whole yeast lysates. Both GLM approaches and the Fisher Exact test performed adequately, each with their unique limitations. We subsequently compared the proteomes of normal tonsil epithelium and HNSCC using this approach and identified 86 proteins with differential spectral counts between normal tonsil epithelium and HNSCC. We selected 18 proteins from this comparison for verification of protein levels between the individual normal and tumor tissues using liquid chromatography−multiple reaction monitoring mass spectrometry (LC−MRM-MS). This analysis confirmed the magnitude and direction of the protein expression differences in all 6 proteins for which reliable data could be obtained. Our analysis demonstrates that shotgun proteomic data sets from different tissue phenotypes are sufficiently rich in quantitative information and that statistically significant differences in proteins spectral counts reflect the underlying biology of the samples. PMID:20586475

Statistical Methods for Proteomic Biomarker Discovery based on Feature Extraction or Functional Modeling Approaches.

PubMed

Morris, Jeffrey S

2012-01-01

In recent years, developments in molecular biotechnology have led to the increased promise of detecting and validating biomarkers, or molecular markers that relate to various biological or medical outcomes. Proteomics, the direct study of proteins in biological samples, plays an important role in the biomarker discovery process. These technologies produce complex, high dimensional functional and image data that present many analytical challenges that must be addressed properly for effective comparative proteomics studies that can yield potential biomarkers. Specific challenges include experimental design, preprocessing, feature extraction, and statistical analysis accounting for the inherent multiple testing issues. This paper reviews various computational aspects of comparative proteomic studies, and summarizes contributions I along with numerous collaborators have made. First, there is an overview of comparative proteomics technologies, followed by a discussion of important experimental design and preprocessing issues that must be considered before statistical analysis can be done. Next, the two key approaches to analyzing proteomics data, feature extraction and functional modeling, are described. Feature extraction involves detection and quantification of discrete features like peaks or spots that theoretically correspond to different proteins in the sample. After an overview of the feature extraction approach, specific methods for mass spectrometry ( Cromwell ) and 2D gel electrophoresis ( Pinnacle ) are described. The functional modeling approach involves modeling the proteomic data in their entirety as functions or images. A general discussion of the approach is followed by the presentation of a specific method that can be applied, wavelet-based functional mixed models, and its extensions. All methods are illustrated by application to two example proteomic data sets, one from mass spectrometry and one from 2D gel electrophoresis. While the specific methods presented are applied to two specific proteomic technologies, MALDI-TOF and 2D gel electrophoresis, these methods and the other principles discussed in the paper apply much more broadly to other expression proteomics technologies.

Proteomics: a new approach to the study of disease.

PubMed

Chambers, G; Lawrie, L; Cash, P; Murray, G I

2000-11-01

The global analysis of cellular proteins has recently been termed proteomics and is a key area of research that is developing in the post-genome era. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. The application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets. This review aims to explain briefly the background to proteomics and then to outline proteomic techniques. Applications to the study of human disease conditions ranging from cancer to infectious diseases are reviewed. Finally, possible future advances are briefly considered, especially those which may lead to faster sample throughput and increased sensitivity for the detection of individual proteins. Copyright 2000 John Wiley & Sons, Ltd.

Clinical proteomic analysis of scrub typhus infection.

PubMed

Park, Edmond Changkyun; Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Hayoung; Song, Hyun Seok; Jun, Sangmi; Kim, Gun-Hwa; Lee, Chang-Seop; Kim, Seung Il

2018-01-01

Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi , and normal expression was largely rescued by antibiotic treatment. This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression

PubMed Central

Catusse, Julie; Strub, Jean-Marc; Job, Claudette; Van Dorsselaer, Alain; Job, Dominique

2008-01-01

Proteomic analysis of mature sugarbeet seeds led to the identification of 759 proteins and their specific tissue expression in root, cotyledons, and perisperm. In particular, the proteome of the perispermic storage tissue found in many seeds of the Caryophyllales is described here. The data allowed us to reconstruct in detail the metabolism of the seeds toward recapitulating facets of seed development and provided insights into complex behaviors such as germination. The seed appears to be well prepared to mobilize the major classes of reserves (the proteins, triglycerides, phytate, and starch) during germination, indicating that the preparation of the seed for germination is mainly achieved during its maturation on the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic trait defined as the potential to produce vigorous seedlings, such as glycine betaine accumulation in seeds. This study also identified several proteins that, to our knowledge, have not previously been described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by a cap-independent process. The study of the tissue specificity of the seed proteome demonstrated a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, indicating a division of metabolic tasks between the various tissues. Furthermore, the perisperm, although it is known as a dead tissue, appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo. PMID:18635686

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

PubMed

Li, Xiao-jun; Yi, Eugene C; Kemp, Christopher J; Zhang, Hui; Aebersold, Ruedi

2005-09-01

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

Proteomics data repositories

PubMed Central

Riffle, Michael; Eng, Jimmy K.

2010-01-01

The field of proteomics, particularly the application of mass spectrometry analysis to protein samples, is well-established and growing rapidly. Proteomics studies generate large volumes of raw experimental data and inferred biological results. To facilitate the dissemination of these data, centralized data repositories have been developed that make the data and results accessible to proteomics researchers and biologists alike. This review of proteomics data repositories focuses exclusively on freely-available, centralized data resources that disseminate or store experimental mass spectrometry data and results. The resources chosen reflect a current “snapshot” of the state of resources available with an emphasis placed on resources that may be of particular interest to yeast researchers. Resources are described in terms of their intended purpose and the features and functionality provided to users. PMID:19795424

A simple dual online ultra-high pressure liquid chromatography system (sDO-UHPLC) for high throughput proteome analysis.

PubMed

Lee, Hangyeore; Mun, Dong-Gi; Bae, Jingi; Kim, Hokeun; Oh, Se Yeon; Park, Young Soo; Lee, Jae-Hyuk; Lee, Sang-Won

2015-08-21

We report a new and simple design of a fully automated dual-online ultra-high pressure liquid chromatography system. The system employs only two nano-volume switching valves (a two-position four port valve and a two-position ten port valve) that direct solvent flows from two binary nano-pumps for parallel operation of two analytical columns and two solid phase extraction (SPE) columns. Despite the simple design, the sDO-UHPLC offers many advantageous features that include high duty cycle, back flushing sample injection for fast and narrow zone sample injection, online desalting, high separation resolution and high intra/inter-column reproducibility. This system was applied to analyze proteome samples not only in high throughput deep proteome profiling experiments but also in high throughput MRM experiments.

Quantitative proteomics in cardiovascular research: global and targeted strategies

PubMed Central

Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun

2014-01-01

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501

Science, marketing and wishful thinking in quantitative proteomics.

PubMed

Hackett, Murray

2008-11-01

In a recent editorial (J. Proteome Res. 2007, 6, 1633) and elsewhere questions have been raised regarding the lack of attention paid to good analytical practice with respect to the reporting of quantitative results in proteomics. Using those comments as a starting point, several issues are discussed that relate to the challenges involved in achieving adequate sampling with MS-based methods in order to generate valid data for large-scale studies. The discussion touches on the relationships that connect sampling depth and the power to detect protein abundance change, conflict of interest, and strategies to overcome bureaucratic obstacles that impede the use of peer-to-peer technologies for transfer and storage of large data files generated in such experiments.

Using Public Data for Comparative Proteome Analysis in Precision Medicine Programs.

PubMed

Hughes, Christopher S; Morin, Gregg B

2018-03-01

Maximizing the clinical utility of information obtained in longitudinal precision medicine programs would benefit from robust comparative analyses to known information to assess biological features of patient material toward identifying the underlying features driving their disease phenotype. Herein, the potential for utilizing publically deposited mass-spectrometry-based proteomics data to perform inter-study comparisons of cell-line or tumor-tissue materials is investigated. To investigate the robustness of comparison between MS-based proteomics studies carried out with different methodologies, deposited data representative of label-free (MS1) and isobaric tagging (MS2 and MS3 quantification) are utilized. In-depth quantitative proteomics data acquired from analysis of ovarian cancer cell lines revealed the robust recapitulation of observable gene expression dynamics between individual studies carried out using significantly different methodologies. The observed signatures enable robust inter-study clustering of cell line samples. In addition, the ability to classify and cluster tumor samples based on observed gene expression trends when using a single patient sample is established. With this analysis, relevant gene expression dynamics are obtained from a single patient tumor, in the context of a precision medicine analysis, by leveraging a large cohort of repository data as a comparator. Together, these data establish the potential for state-of-the-art MS-based proteomics data to serve as resources for robust comparative analyses in precision medicine applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

PubMed Central

Sousa, Josane F.; Ham, Amy-Joan L.; Whitwell, Corbin; Nam, Ki Taek; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Zhang, Bing; Li, Ming; LaFleur, Bonnie; Liebler, Daniel C.; Goldenring, James R.

2013-01-01

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

NASA Astrophysics Data System (ADS)

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-06-01

Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.

Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

PubMed Central

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-01-01

Mass spectrometry–based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications. PMID:27049631

Trypsin and MALDI matrix pre-coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

PubMed Central

Zubair, Faizan; Laibinis, Paul E.; Swisher, William G.; Yang, Junhai; Spraggins, Jeffrey M.; Norris, Jeremy L.; Caprioli, Richard M.

2017-01-01

Prefabricated surfaces containing α-cyano-4-hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α-cyano-4-hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography-tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine-rich C-kinase substrate (29.8 kDa) and spectrin alpha chain, non-erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre-coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. PMID:27676701

Marine proteomics: a critical assessment of an emerging technology.

PubMed

Slattery, Marc; Ankisetty, Sridevi; Corrales, Jone; Marsh-Hunkin, K Erica; Gochfeld, Deborah J; Willett, Kristine L; Rimoldi, John M

2012-10-26

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives.

PubMed

Zhou, Li; Wang, Kui; Li, Qifu; Nice, Edouard C; Zhang, Haiyuan; Huang, Canhua

2016-01-01

Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy.

Highly efficient proteome analysis with combination of protein pre-fractionation by preparative microscale solution isoelectric focusing and identification by μRPLC-MS/MS with serially coupled long microcolumn.

PubMed

Tao, Dingyin; Sun, Liangliang; Zhu, Guijie; Liang, Yu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-01-01

To improve the efficiency of proteome analysis, a strategy with the combination of protein pre-fractionation by preparative microscale solution isoelectric focusing, peptide separation by μRPLC with serially coupled long microcolumn and protein identification by ESI-MS/MS was proposed. By preparative microscale solution isoelectric focusing technique, proteins extracted from whole cell lysates of Escherichia coli were fractionated into five chambers divided by isoelectric membranes, respectively with pH range from 3.0 to 4.6, 4.6 to 5.4, 5.4 to 6.2, 6.2 to 7.0 and 7.0 to 10.0. Compared to the traditional on-gel IFF, the protein recovery could be obviously improved to over 95%. Subsequently, the enriched and fractionated proteins in each chamber were digested, and further separated by a 30-cm long serially coupled RP microcolumn. Through the detection by ESI-MS/MS, about 200 proteins were identified in each fraction, and in total 835 proteins were identified even with one-dimensional μRPLC-MS/MS system. All these results demonstrate that by such a combination strategy, highly efficient proteome analysis could be achieved, not only due to the in-solution protein enrichment and pre-fractionation with improved protein recovery but also owing to the increased separation capacity of serially coupled long μRPLC columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis*

PubMed Central

Gillet, Ludovic C.; Navarro, Pedro; Tate, Stephen; Röst, Hannes; Selevsek, Nathalie; Reiter, Lukas; Bonner, Ron; Aebersold, Ruedi

2012-01-01

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400–1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics. PMID:22261725

Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens

PubMed Central

Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

2015-01-01

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3–16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3–16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis. PMID:26580073

Proteomic and Properties Analysis of Botanical Insecticide Rhodojaponin III-Induced Response of the Diamondback Moth, Plutella xyllostella (L.)

PubMed Central

Dong, Xiaolin; Zhai, Yifan; Hu, Meiying; Zhong, Guohua; Huang, Wanjun; Zheng, Zhihua; Han, Pengfei

2013-01-01

Background Rhodojaponin III, as a botanical insecticide, affects a wide variety of biological processes in insects, including reduction of feeding, suspension of development, and oviposition deterring of adults in a dose-dependent manner. However, the mode of these actions remains obscure. Principal Findings In this study, a comparative proteomic approach was adopted to examine the effect of rhodojaponin III on the Plutella xyllostella (L.). Following treating 48 hours, newly emergence moths were collected and protein samples were prepared. The proteins were separated by 2-DE, and total 31 proteins were significantly affected by rhodojaponin III compared to the control identified by MALDI-TOF/TOF-MS/MS. These differentially expressed proteins act in the nervous transduction, odorant degradation and metabolic change pathways. Further, gene expression patterns in treated and untreated moths were confirmed by qRT-PCR and western blot analysis. RNAi of the chemosensory protein (PxCSP) gene resulted in oviposition significantly increased on cabbage plants treated with rhodojaponin III. Conclusions These rhodojaponin III-induced proteins and gene properties analysis would be essential for a better understanding of the potential molecular mechanism of the response to rhodojaponin III from moths of P. xylostella. PMID:23861792

Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach.

PubMed

Santa, Cátia; Anjo, Sandra I; Manadas, Bruno

2016-07-01

Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest, especially because of the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work, two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields were compared with the results from the same sample without precipitation. From this study, it was possible to conclude that in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery and number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone compared with the original sample. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Robust Two-Dimensional Separation of Intact Proteins for Bottom-Up Tandem Mass Spectrometry of the Human CSF Proteome

PubMed Central

Bora, Adriana; Anderson, Carol; Bachani, Muznabanu; Nath, Avindra; Cotter, Robert J.

2012-01-01

The cerebrospinal fluid (CSF) is produced in the brain by cells in the choroid plexus at a rate of 500mL/day. It is the only body fluid in direct contact with the brain. Thus, any changes in the CSF composition will reflect pathological processes and make CSF a potential source of biomarkers for different disease states. Proteomics offers a comprehensive view of the proteins found in CSF. In this study, we use a recently developed non-gel based method of sample preparation of CSF followed by liquid chromatography high accuracy mass spectrometry (LC-MS) for MS and MS/MS analyses, allowing unambiguous identification of peptides/proteins. Gel-eluted liquid fraction entrapment electrophoresis (Gelfree) is used to separate a CSF complex protein mixture in 12 user-selectable liquid-phase molecular weight fractions. Using this high throughput workflow we have been able to separate CSF intact proteins over a broad mass range 3.5 kDa-100 kDa with high resolution between 15 kDa and 100 kDa in 2 hours and 40 min. We have completely eliminated albumin and were able to interrogate the low abundance CSF proteins in a highly reproducible manner from different CSF samples in the same time. Using LC-MS as a downstream analysis, we identified 368 proteins using MidiTrap G-10 desalting columns and 166 proteins (including 57 unique proteins) using Zeba spin columns with 5% false discovery rate (FDR). Prostaglandin D2 synthase, Chromogranin A, Apolipoprotein E, Chromogranin B, Secretogranin III, Cystatin C, VGF nerve growth factor, Cadherin 2 are a few of the proteins that were characterized. The Gelfree-LC-MS is a robust method for the analysis of the human proteome that we will use to develop biomarkers for several neurodegenerative diseases and to quantitate these markers using multiple reaction monitoring. PMID:22537003

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

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

YPED: An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

PubMed Central

Colangelo, Christopher M.; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L.; Carriero, Nicholas J.; Gulcicek, Erol E.; Lam, TuKiet T.; Wu, Terence; Bjornson, Robert D.; Bruce, Can; Nairn, Angus C.; Rinehart, Jesse; Miller, Perry L.; Williams, Kenneth R.

2015-01-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry (LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED’s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. PMID:25712262

The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis.

PubMed

Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

2016-09-01

The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

PubMed

Colangelo, Christopher M; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L; Carriero, Nicholas J; Gulcicek, Erol E; Lam, TuKiet T; Wu, Terence; Bjornson, Robert D; Bruce, Can; Nairn, Angus C; Rinehart, Jesse; Miller, Perry L; Williams, Kenneth R

2015-02-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature.

PubMed

Jung, Hye Jin

2017-01-01

Cancer stem cells (CSCs) have been proposed as central drivers of tumor initiation, progression, recurrence, and therapeutic resistance. Therefore, identifying stem-like cells within cancers and understanding their properties is crucial for the development of effective anticancer therapies. Recently, chemical proteomics has become a powerful tool to efficiently determine protein networks responsible for CSC pathophysiology and comprehensively elucidate molecular mechanisms of drug action against CSCs. This review provides an overview of major methodologies utilized in chemical proteomic approaches. In addition, recent successful chemical proteomic applications targeting CSCs are highlighted. Future direction of potential CSC research by integrating chemical genomic and proteomic data obtained from a single biological sample of CSCs are also suggested in this review. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Head and neck cancer: proteomic advances and biomarker achievements.

PubMed

Rezende, Taia Maria Berto; de Souza Freire, Mirna; Franco, Octávio Luiz

2010-11-01

Tumors of the head and neck comprise an important neoplasia group, the incidence of which is increasing in many parts of the world. Recent advances in diagnostic and therapeutic techniques for these lesions have yielded novel molecular targets, uncovered signal pathway dominance, and advanced early cancer detection. Proteomics is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the analysis of different types of samples. The proteomic profiles of different types of cancer have been studied, and this has provided remarkable advances in cancer understanding. This review covers recent advances for head and neck cancer; it encompasses the risk factors, pathogenesis, proteomic tools that can help in understanding cancer, and new proteomic findings in this type of cancer. Copyright © 2010 American Cancer Society.

Trends in mass spectrometry instrumentation for proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Richard D.

2002-12-01

Mass spectrometry has become a primary tool for proteomics due to its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the needs for increased capabilities for proteome measurements are immense and are now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements, and promise more than order of magnitude improvements in sensitivity, dynamic range, and throughput for proteomic analyses in themore » near future.« less

Molecular Profiles for Lung Cancer Pathogenesis and Detection in US Veterans

DTIC Science & Technology

2012-10-01

will be further strengthened via Multiple Reaction Monitoring ( MRM ) performed on the remaining samples by the Vanderbilt group. MRM using mass...proteomics detects all protein changes in the sample in an unfocused fashion, MRM is targeted and highly selective, allowing us to specifically look for...proteins of interest. To this end, we have generated a list of candidate proteins for MRM utilizing shotgun proteomic, mRNA array, and miRNA array

Proteomic Workflows for Biomarker Identification Using Mass Spectrometry — Technical and Statistical Considerations during Initial Discovery

PubMed Central

Orton, Dennis J.; Doucette, Alan A.

2013-01-01

Identification of biomarkers capable of differentiating between pathophysiological states of an individual is a laudable goal in the field of proteomics. Protein biomarker discovery generally employs high throughput sample characterization by mass spectrometry (MS), being capable of identifying and quantifying thousands of proteins per sample. While MS-based technologies have rapidly matured, the identification of truly informative biomarkers remains elusive, with only a handful of clinically applicable tests stemming from proteomic workflows. This underlying lack of progress is attributed in large part to erroneous experimental design, biased sample handling, as well as improper statistical analysis of the resulting data. This review will discuss in detail the importance of experimental design and provide some insight into the overall workflow required for biomarker identification experiments. Proper balance between the degree of biological vs. technical replication is required for confident biomarker identification. PMID:28250400

Halogenated peptides as internal standards (H-PINS): introduction of an MS-based internal standard set for liquid chromatography-mass spectrometry.

PubMed

Mirzaei, Hamid; Brusniak, Mi-Youn; Mueller, Lukas N; Letarte, Simon; Watts, Julian D; Aebersold, Ruedi

2009-08-01

As the application for quantitative proteomics in the life sciences has grown in recent years, so has the need for more robust and generally applicable methods for quality control and calibration. The reliability of quantitative proteomics is tightly linked to the reproducibility and stability of the analytical platforms, which are typically multicomponent (e.g. sample preparation, multistep separations, and mass spectrometry) with individual components contributing unequally to the overall system reproducibility. Variations in quantitative accuracy are thus inevitable, and quality control and calibration become essential for the assessment of the quality of the analyses themselves. Toward this end, the use of internal standards cannot only assist in the detection and removal of outlier data acquired by an irreproducible system (quality control) but can also be used for detection of changes in instruments for their subsequent performance and calibration. Here we introduce a set of halogenated peptides as internal standards. The peptides are custom designed to have properties suitable for various quality control assessments, data calibration, and normalization processes. The unique isotope distribution of halogenated peptides makes their mass spectral detection easy and unambiguous when spiked into complex peptide mixtures. In addition, they were designed to elute sequentially over an entire aqueous to organic LC gradient and to have m/z values within the commonly scanned mass range (300-1800 Da). In a series of experiments in which these peptides were spiked into an enriched N-glycosite peptide fraction (i.e. from formerly N-glycosylated intact proteins in their deglycosylated form) isolated from human plasma, we show the utility and performance of these halogenated peptides for sample preparation and LC injection quality control as well as for retention time and mass calibration. Further use of the peptides for signal intensity normalization and retention time synchronization for selected reaction monitoring experiments is also demonstrated.

Halogenated Peptides as Internal Standards (H-PINS)

PubMed Central

Mirzaei, Hamid; Brusniak, Mi-Youn; Mueller, Lukas N.; Letarte, Simon; Watts, Julian D.; Aebersold, Ruedi

2009-01-01

As the application for quantitative proteomics in the life sciences has grown in recent years, so has the need for more robust and generally applicable methods for quality control and calibration. The reliability of quantitative proteomics is tightly linked to the reproducibility and stability of the analytical platforms, which are typically multicomponent (e.g. sample preparation, multistep separations, and mass spectrometry) with individual components contributing unequally to the overall system reproducibility. Variations in quantitative accuracy are thus inevitable, and quality control and calibration become essential for the assessment of the quality of the analyses themselves. Toward this end, the use of internal standards cannot only assist in the detection and removal of outlier data acquired by an irreproducible system (quality control) but can also be used for detection of changes in instruments for their subsequent performance and calibration. Here we introduce a set of halogenated peptides as internal standards. The peptides are custom designed to have properties suitable for various quality control assessments, data calibration, and normalization processes. The unique isotope distribution of halogenated peptides makes their mass spectral detection easy and unambiguous when spiked into complex peptide mixtures. In addition, they were designed to elute sequentially over an entire aqueous to organic LC gradient and to have m/z values within the commonly scanned mass range (300–1800 Da). In a series of experiments in which these peptides were spiked into an enriched N-glycosite peptide fraction (i.e. from formerly N-glycosylated intact proteins in their deglycosylated form) isolated from human plasma, we show the utility and performance of these halogenated peptides for sample preparation and LC injection quality control as well as for retention time and mass calibration. Further use of the peptides for signal intensity normalization and retention time synchronization for selected reaction monitoring experiments is also demonstrated. PMID:19411281

Assessing Amide Proton Transfer (APT) MRI Contrast Origins in 9 L Gliosarcoma in the Rat Brain Using Proteomic Analysis.

PubMed

Yan, Kun; Fu, Zongming; Yang, Chen; Zhang, Kai; Jiang, Shanshan; Lee, Dong-Hoon; Heo, Hye-Young; Zhang, Yi; Cole, Robert N; Van Eyk, Jennifer E; Zhou, Jinyuan

2015-08-01

To investigate the biochemical origin of the amide photon transfer (APT)-weighted hyperintensity in brain tumors. Seven 9 L gliosarcoma-bearing rats were imaged at 4.7 T. Tumor and normal brain tissue samples of equal volumes were prepared with a coronal rat brain matrix and a tissue biopsy punch. The total tissue protein and the cytosolic subproteome were extracted from both samples. Protein samples were analyzed using two-dimensional gel electrophoresis, and the proteins with significant abundance changes were identified by mass spectrometry. There was a significant increase in the cytosolic protein concentration in the tumor, compared to normal brain regions, but the total protein concentrations were comparable. The protein profiles of the tumor and normal brain tissue differed significantly. Six cytosolic proteins, four endoplasmic reticulum proteins, and five secreted proteins were considerably upregulated in the tumor. Our experiments confirmed an increase in the cytosolic protein concentration in tumors and identified several key proteins that may cause APT-weighted hyperintensity.

A HUPO test sample study reveals common problems in mass spectrometry-based proteomics

PubMed Central

Bell, Alexander W.; Deutsch, Eric W.; Au, Catherine E.; Kearney, Robert E.; Beavis, Ron; Sechi, Salvatore; Nilsson, Tommy; Bergeron, John J.M.

2009-01-01

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics. PMID:19448641

Selected reaction monitoring mass spectrometry: a methodology overview.

PubMed

Ebhardt, H Alexander

2014-01-01

Moving past the discovery phase of proteomics, the term targeted proteomics combines multiple approaches investigating a certain set of proteins in more detail. One such targeted proteomics approach is the combination of liquid chromatography and selected or multiple reaction monitoring mass spectrometry (SRM, MRM). SRM-MS requires prior knowledge of the fragmentation pattern of peptides, as the presence of the analyte in a sample is determined by measuring the m/z values of predefined precursor and fragment ions. Using scheduled SRM-MS, many analytes can robustly be monitored allowing for high-throughput sample analysis of the same set of proteins over many conditions. In this chapter, fundaments of SRM-MS are explained as well as an optimized SRM pipeline from assay generation to data analyzed.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing.

PubMed

Polati, Rita; Menini, Michele; Robotti, Elisa; Millioni, Renato; Marengo, Emilio; Novelli, Enrico; Balzan, Stefania; Cecconi, Daniela

2012-12-01

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

PubMed

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Monitoring Peptidase Activities in Complex Proteomes by MALDI-TOF Mass Spectrometry

PubMed Central

Villanueva, Josep; Nazarian, Arpi; Lawlor, Kevin; Tempst, Paul

2009-01-01

Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semi-quantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide-substrates using robotic extraction and a MALDI-TOF mass spectrometric read-out. Relative quantitation of the peptide metabolites is done by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be employed for diagnostic or predictive purposes and enables profiling of 96 samples in 30 hours. It could be tailored to many diagnostic and pharmaco-dynamic purposes, as a read-out of catalytic and metabolic activities in body fluids or tissues. PMID:19617888

Virtual Labs in proteomics: new E-learning tools.

PubMed

Ray, Sandipan; Koshy, Nicole Rachel; Reddy, Panga Jaipal; Srivastava, Sanjeeva

2012-05-17

Web-based educational resources have gained enormous popularity recently and are increasingly becoming a part of modern educational systems. Virtual Labs are E-learning platforms where learners can gain the experience of practical experimentation without any direct physical involvement on real bench work. They use computerized simulations, models, videos, animations and other instructional technologies to create interactive content. Proteomics being one of the most rapidly growing fields of the biological sciences is now an important part of college and university curriculums. Consequently, many E-learning programs have started incorporating the theoretical and practical aspects of different proteomic techniques as an element of their course work in the form of Video Lectures and Virtual Labs. To this end, recently we have developed a Virtual Proteomics Lab at the Indian Institute of Technology Bombay, which demonstrates different proteomics techniques, including basic and advanced gel and MS-based protein separation and identification techniques, bioinformatics tools and molecular docking methods, and their applications in different biological samples. This Tutorial will discuss the prominent Virtual Labs featuring proteomics content, including the Virtual Proteomics Lab of IIT-Bombay, and E-resources available for proteomics study that are striving to make proteomic techniques and concepts available and accessible to the student and research community. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 14). Details can be found at: http://www.proteomicstutorials.org/. Copyright © 2012 Elsevier B.V. All rights reserved.

Construction of a nasopharyngeal carcinoma 2D/MS repository with Open Source XML database--Xindice.

PubMed

Li, Feng; Li, Maoyu; Xiao, Zhiqiang; Zhang, Pengfei; Li, Jianling; Chen, Zhuchu

2006-01-11

Many proteomics initiatives require integration of all information with uniformcriteria from collection of samples and data display to publication of experimental results. The integration and exchanging of these data of different formats and structure imposes a great challenge to us. The XML technology presents a promise in handling this task due to its simplicity and flexibility. Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Southeast Asia, which has marked geographic and racial differences in incidence. Although there are some cancer proteome databases now, there is still no NPC proteome database. The raw NPC proteome experiment data were captured into one XML document with Human Proteome Markup Language (HUP-ML) editor and imported into native XML database Xindice. The 2D/MS repository of NPC proteome was constructed with Apache, PHP and Xindice to provide access to the database via Internet. On our website, two methods, keyword query and click query, were provided at the same time to access the entries of the NPC proteome database. Our 2D/MS repository can be used to share the raw NPC proteomics data that are generated from gel-based proteomics experiments. The database, as well as the PHP source codes for constructing users' own proteome repository, can be accessed at http://www.xyproteomics.org/.

Detergents: Friends not foes for high-performance membrane proteomics toward precision medicine.

PubMed

Zhang, Xi

2017-02-01

Precision medicine, particularly therapeutics, emphasizes the atomic-precise, dynamic, and systems visualization of human membrane proteins and their endogenous modifiers. For years, bottom-up proteomics has grappled with removing and avoiding detergents, yet faltered at the therapeutic-pivotal membrane proteins, which have been tackled by classical approaches and are known for decades refractory to single-phase aqueous or organic denaturants. Hydrophobicity and aggregation commonly challenge tissue and cell lysates, biofluids, and enriched samples. Frequently, expected membrane proteins and peptides are not identified by shotgun bottom-up proteomics, let alone robust quantitation. This review argues the cause of this proteomic crisis is not detergents per se, but the choice of detergents. Recently, inclusion of compatible detergents for membrane protein extraction and digestion has revealed stark improvements in both quantitative and structural proteomics. This review analyzes detergent properties behind recent proteomic advances, and proposes that rational use of detergents may reconcile outstanding membrane proteomics dilemmas, enabling ultradeep coverage and minimal artifacts for robust protein and endogenous PTM measurements. The simplicity of detergent tools confers bottom-up membrane proteomics the sophistication toward precision medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hospitalized Premature Infants Are Colonized by Related Bacterial Strains with Distinct Proteomic Profiles

PubMed Central

Xiong, Weili; Olm, Matthew R.; Thomas, Brian C.; Baker, Robyn; Firek, Brian; Morowitz, Michael J.; Hettich, Robert L.

2018-01-01

ABSTRACT During the first weeks of life, microbial colonization of the gut impacts human immune system maturation and other developmental processes. In premature infants, aberrant colonization has been implicated in the onset of necrotizing enterocolitis (NEC), a life-threatening intestinal disease. To study the premature infant gut colonization process, genome-resolved metagenomics was conducted on 343 fecal samples collected during the first 3 months of life from 35 premature infants housed in a neonatal intensive care unit, 14 of whom developed NEC, and metaproteomic measurements were made on 87 samples. Microbial community composition and proteomic profiles remained relatively stable on the time scale of a week, but the proteome was more variable. Although genetically similar organisms colonized many infants, most infants were colonized by distinct strains with metabolic profiles that could be distinguished using metaproteomics. Microbiome composition correlated with infant, antibiotics administration, and NEC diagnosis. Communities were found to cluster into seven primary types, and community type switched within infants, sometimes multiple times. Interestingly, some communities sampled from the same infant at subsequent time points clustered with those of other infants. In some cases, switches preceded onset of NEC; however, no species or community type could account for NEC across the majority of infants. In addition to a correlation of protein abundances with organism replication rates, we found that organism proteomes correlated with overall community composition. Thus, this genome-resolved proteomics study demonstrated that the contributions of individual organisms to microbiome development depend on microbial community context. PMID:29636439

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

PubMed

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

PubMed

Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

PubMed Central

Russ, Holger A.; Landsman, Limor; Moss, Christopher L.; Higdon, Roger; Greer, Renee L.; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

2016-01-01

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

Enrichment and proteomic analysis of plasma membrane from rat dorsal root ganglions

PubMed Central

2009-01-01

Background Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. Plasma membrane (PM) of DRG cells plays important roles in their functions. PM proteins are main performers of the functions. However, mainly due to the very low amount of DRG that leads to the difficulties in PM sample collection, few proteomic analyses on the PM have been reported and it is a subject that demands further investigation. Results By using aqueous polymer two-phase partition in combination with high salt and high pH washing, PMs were efficiently enriched, demonstrated by western blot analysis. A total of 954 non-redundant proteins were identified from the plasma membrane-enriched preparation with CapLC-MS/MS analysis subsequent to protein separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or shotgun digestion. 205 (21.5%) of the identified proteins were unambiguously assigned as PM proteins, including a large number of signal proteins, receptors, ion channel and transporters. Conclusion The aqueous polymer two-phase partition is a simple, rapid and relatively inexpensive method. It is well suitable for the purification of PMs from small amount of tissues. Therefore, it is reasonable for the DRG PM to be enriched by using aqueous two-phase partition as a preferred method. Proteomic analysis showed that DRG PM was rich in proteins involved in the fundamental biological processes including material exchange, energy transformation and information transmission, etc. These data would help to our further understanding of the fundamental DRG functions. PMID:19889238

The Magellania venosa Biomineralizing Proteome: A Window into Brachiopod Shell Evolution

PubMed Central

Jackson, Daniel J.; Mann, Karlheinz; Häussermann, Vreni; Schilhabel, Markus B.; Lüter, Carsten; Griesshaber, Erika; Schmahl, Wolfgang; Wörheide, Gert

2015-01-01

Brachiopods are a lineage of invertebrates well known for the breadth and depth of their fossil record. Although the quality of this fossil record attracts the attention of paleontologists, geochemists, and paleoclimatologists, modern day brachiopods are also of interest to evolutionary biologists due to their potential to address a variety of questions ranging from developmental biology to biomineralization. The brachiopod shell is a composite material primarily composed of either calcite or calcium phosphate in close association with proteins and polysaccharides which give these composite structures their material properties. The information content of these biomolecules, sequestered within the shell during its construction, has the potential to inform hypotheses focused on describing how brachiopod shell formation evolved. Here, using high throughput proteomic approaches and next generation sequencing, we have surveyed and characterized the first shell-proteome and shell-forming transcriptome of any brachiopod, the South American Magellania venosa (Rhynchonelliformea: Terebratulida). We find that the seven most abundant proteins present in the shell are unique to M. venosa, but that these proteins display biochemical features found in other metazoan biomineralization proteins. We can also detect some M. venosa proteins that display significant sequence similarity to other metazoan biomineralization proteins, suggesting that some elements of the brachiopod shell-forming proteome are deeply evolutionarily conserved. We also employed a variety of preparation methods to isolate shell proteins and find that in comparison to the shells of other spiralian invertebrates (such as mollusks) the shell ultrastructure of M. venosa may explain the effects these preparation strategies have on our results. PMID:25912046

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

PubMed

Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

2017-08-01

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

PubMed Central

Cilia, M.; Fish, T.; Yang, X.; Mclaughlin, M.; Thannhauser, T. W.

2009-01-01

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques. PMID:19721822

Vintage venoms: proteomic and pharmacological stability of snake venoms stored for up to eight decades.

PubMed

Jesupret, Clémence; Baumann, Kate; Jackson, Timothy N W; Ali, Syed Abid; Yang, Daryl C; Greisman, Laura; Kern, Larissa; Steuten, Jessica; Jouiaei, Mahdokht; Casewell, Nicholas R; Undheim, Eivind A B; Koludarov, Ivan; Debono, Jordan; Low, Dolyce H W; Rossi, Sarah; Panagides, Nadya; Winter, Kelly; Ignjatovic, Vera; Summerhayes, Robyn; Jones, Alun; Nouwens, Amanda; Dunstan, Nathan; Hodgson, Wayne C; Winkel, Kenneth D; Monagle, Paul; Fry, Bryan Grieg

2014-06-13

For over a century, venom samples from wild snakes have been collected and stored around the world. However, the quality of storage conditions for "vintage" venoms has rarely been assessed. The goal of this study was to determine whether such historical venom samples are still biochemically and pharmacologically viable for research purposes, or if new sample efforts are needed. In total, 52 samples spanning 5 genera and 13 species with regional variants of some species (e.g., 14 different populations of Notechis scutatus) were analysed by a combined proteomic and pharmacological approach to determine protein structural stability and bioactivity. When venoms were not exposed to air during storage, the proteomic results were virtually indistinguishable from that of fresh venom and bioactivity was equivalent or only slightly reduced. By contrast, a sample of Acanthophis antarcticus venom that was exposed to air (due to a loss of integrity of the rubber stopper) suffered significant degradation as evidenced by the proteomics profile. Interestingly, the neurotoxicity of this sample was nearly the same as fresh venom, indicating that degradation may have occurred in the free N- or C-terminus chains of the proteins, rather than at the tips of loops where the functional residues are located. These results suggest that these and other vintage venom collections may be of continuing value in toxin research. This is particularly important as many snake species worldwide are declining due to habitat destruction or modification. For some venoms (such as N. scutatus from Babel Island, Flinders Island, King Island and St. Francis Island) these were the first analyses ever conducted and these vintage samples may represent the only venom ever collected from these unique island forms of tiger snakes. Such vintage venoms may therefore represent the last remaining stocks of some local populations and thus are precious resources. These venoms also have significant historical value as the Oxyuranus venoms analysed include samples from the first coastal taipan (Oxyuranus scutellatus) collected for antivenom production (the snake that killed the collector Kevin Budden), as well as samples from the first Oxyuranus microlepidotus specimen collected after the species' rediscovery in 1976. These results demonstrate that with proper storage techniques, venom samples can retain structural and pharmacological stability. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins.

PubMed

Brick, Tabea; Ege, Markus; Boeren, Sjef; Böck, Andreas; von Mutius, Erika; Vervoort, Jacques; Hettinga, Kasper

2017-08-31

Consumption of raw cow's milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general is missing. The aim of this study was to compare the native milk serum proteome between raw cow's milk and various industrially applied processing methods, i.e., homogenization, fat separation, pasteurization, ultra-heat treatment (UHT), treatment for extended shelf-life (ESL), and conventional boiling. Each processing method was applied to the same three pools of raw milk. Levels of detectable proteins were quantified by liquid chromatography/tandem mass spectrometry following filter aided sample preparation. In total, 364 milk serum proteins were identified. The 140 proteins detectable in 66% of all samples were entered in a hierarchical cluster analysis. The resulting proteomics pattern separated mainly as high (boiling, UHT, ESL) versus no/low heat treatment (raw, skimmed, pasteurized). Comparing these two groups revealed 23 individual proteins significantly reduced by heating, e.g., lactoferrin (log2-fold change = -0.37, p = 0.004), lactoperoxidase (log2-fold change = -0.33, p = 0.001), and lactadherin (log2-fold change = -0.22, p = 0.020). The abundance of these heat sensitive proteins found in higher quantity in native cow's milk compared to heat treated milk, renders them potential candidates for protection from asthma, allergies, and respiratory infections.

Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

PubMed Central

Biesemann, Christoph; Grønborg, Mads; Luquet, Elisa; Wichert, Sven P; Bernard, Véronique; Bungers, Simon R; Cooper, Ben; Varoqueaux, Frédérique; Li, Liyi; Byrne, Jennifer A; Urlaub, Henning; Jahn, Olaf; Brose, Nils; Herzog, Etienne

2014-01-01

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease. PMID:24413018

Unraveling snake venom complexity with 'omics' approaches: challenges and perspectives.

PubMed

Zelanis, André; Tashima, Alexandre Keiji

2014-09-01

The study of snake venom proteomes (venomics) has been experiencing a burst of reports, however the comprehensive knowledge of the dynamic range of proteins present within a single venom, the set of post-translational modifications (PTMs) as well as the lack of a comprehensive database related to venom proteins are among the main challenges in venomics research. The phenotypic plasticity in snake venom proteomes together with their inherent toxin proteoform diversity, points out to the use of integrative analysis in order to better understand their actual complexity. In this regard, such a systems venomics task should encompass the integration of data from transcriptomic and proteomic studies (specially the venom gland proteome), the identification of biological PTMs, and the estimation of artifactual proteomes and peptidomes generated by sample handling procedures. Copyright © 2014 Elsevier Ltd. All rights reserved.

A cost-effective method to get insight into the peritoneal dialysate effluent proteome.

PubMed

Araújo, J E; Jorge, S; Teixeira E Costa, F; Ramos, A; Lodeiro, C; Santos, H M; Capelo, J L

2016-08-11

Protein depletion with acetonitrile and protein equalization with dithiothreitol have been assessed with success as proteomics tools for getting insight into the peritoneal dialysate effluent proteome. The methods proposed are cost-effective, fast and easy of handling, and they match the criteria of analytical minimalism: low sample volume and low reagent consumption. Using two-dimensional gel electrophoresis and peptide mass fingerprinting, a total of 72 unique proteins were identified. Acetonitrile depletes de PDE proteome from high-abundance proteins, such as albumin, and enriches the sample in apolipo-like proteins. Dithiothreitol equalizes the PDE proteome by diminishing the levels of albumin and enriching the extract in immunoglobulin-like proteins. The annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysis, namely that the largest number of proteins lost through peritoneal dialysate are extracellular proteins involved in regulation processes through binding. Renal failure is a growing problem worldwide, and particularly in Europe where the population is getting older. Up-to-date there is a focus of interest in peritoneal dialysis (PD), as it provides a better quality of life and autonomy of the patients than other renal replacement therapies such as haemodialysis. However, PD can only be used during a short period of years, as the peritoneum lost its permeability through time. Therefore to make a breakthrough in PD and consequently contribute to better healthcare system it is urgent to find a group of biomarkers of peritoneum degradation. Here we report on two cost-effective methods for protein depletion in peritoneal dialysate effluent (PDE). The use of ACN and DTT over PDE to deplete high abundant proteins or to equalize the concentration of proteins, respectively, performs well and with similar protein profiles than when the same chemicals are used in human plasma samples. ACN depletes de PDE proteome from large proteins, such as albumin, and enriches the sample in apolipoproteins. DTT equalizes the PDE proteome by diminishing the levels of large proteins such as albumin and enriching the extract in immunoglobulins. Although the number and type of proteins identified are different, the annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysate. Thus, the largest number of proteins lost through peritoneal dialysate belongs to the group of extracellular proteins involved in regulation processes through binding. As for the searching of biomarkers, DTT seems to be the most promising of the two methods because acts as an equalizer and it allows interrogating more proteins in the same sample.

In-depth analysis of protein inference algorithms using multiple search engines and well-defined metrics.

PubMed

Audain, Enrique; Uszkoreit, Julian; Sachsenberg, Timo; Pfeuffer, Julianus; Liang, Xiao; Hermjakob, Henning; Sanchez, Aniel; Eisenacher, Martin; Reinert, Knut; Tabb, David L; Kohlbacher, Oliver; Perez-Riverol, Yasset

2017-01-06

In mass spectrometry-based shotgun proteomics, protein identifications are usually the desired result. However, most of the analytical methods are based on the identification of reliable peptides and not the direct identification of intact proteins. Thus, assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is a critical step in proteomics research. Currently, different protein inference algorithms and tools are available for the proteomics community. Here, we evaluated five software tools for protein inference (PIA, ProteinProphet, Fido, ProteinLP, MSBayesPro) using three popular database search engines: Mascot, X!Tandem, and MS-GF+. All the algorithms were evaluated using a highly customizable KNIME workflow using four different public datasets with varying complexities (different sample preparation, species and analytical instruments). We defined a set of quality control metrics to evaluate the performance of each combination of search engines, protein inference algorithm, and parameters on each dataset. We show that the results for complex samples vary not only regarding the actual numbers of reported protein groups but also concerning the actual composition of groups. Furthermore, the robustness of reported proteins when using databases of differing complexities is strongly dependant on the applied inference algorithm. Finally, merging the identifications of multiple search engines does not necessarily increase the number of reported proteins, but does increase the number of peptides per protein and thus can generally be recommended. Protein inference is one of the major challenges in MS-based proteomics nowadays. Currently, there are a vast number of protein inference algorithms and implementations available for the proteomics community. Protein assembly impacts in the final results of the research, the quantitation values and the final claims in the research manuscript. Even though protein inference is a crucial step in proteomics data analysis, a comprehensive evaluation of the many different inference methods has never been performed. Previously Journal of proteomics has published multiple studies about other benchmark of bioinformatics algorithms (PMID: 26585461; PMID: 22728601) in proteomics studies making clear the importance of those studies for the proteomics community and the journal audience. This manuscript presents a new bioinformatics solution based on the KNIME/OpenMS platform that aims at providing a fair comparison of protein inference algorithms (https://github.com/KNIME-OMICS). Six different algorithms - ProteinProphet, MSBayesPro, ProteinLP, Fido and PIA- were evaluated using the highly customizable workflow on four public datasets with varying complexities. Five popular database search engines Mascot, X!Tandem, MS-GF+ and combinations thereof were evaluated for every protein inference tool. In total >186 proteins lists were analyzed and carefully compare using three metrics for quality assessments of the protein inference results: 1) the numbers of reported proteins, 2) peptides per protein, and the 3) number of uniquely reported proteins per inference method, to address the quality of each inference method. We also examined how many proteins were reported by choosing each combination of search engines, protein inference algorithms and parameters on each dataset. The results show that using 1) PIA or Fido seems to be a good choice when studying the results of the analyzed workflow, regarding not only the reported proteins and the high-quality identifications, but also the required runtime. 2) Merging the identifications of multiple search engines gives almost always more confident results and increases the number of peptides per protein group. 3) The usage of databases containing not only the canonical, but also known isoforms of proteins has a small impact on the number of reported proteins. The detection of specific isoforms could, concerning the question behind the study, compensate for slightly shorter reports using the parsimonious reports. 4) The current workflow can be easily extended to support new algorithms and search engine combinations. Copyright © 2016. Published by Elsevier B.V.

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

PubMed Central

2011-01-01

Background Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology. Result We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site. This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser. Conclusions Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html PMID:21414234

The “Dark Side” of Food Stuff Proteomics: The CPLL-Marshals Investigate

PubMed Central

Righetti, Pier Giorgio; Fasoli, Elisa; D’Amato, Alfonsina; Boschetti, Egisto

2014-01-01

The present review deals with analysis of the proteome of animal and plant-derived food stuff, as well as of non-alcoholic and alcoholic beverages. The survey is limited to those systems investigated with the help of combinatorial peptide ligand libraries, a most powerful technique allowing access to low- to very-low-abundance proteins, i.e., to those proteins that might characterize univocally a given biological system and, in the case of commercial food preparations, attest their genuineness or adulteration. Among animal foods the analysis of cow’s and donkey’s milk is reported, together with the proteomic composition of egg white and yolk, as well as of honey, considered as a hybrid between floral and animal origin. In terms of plant and fruits, a survey is offered of spinach, artichoke, banana, avocado, mango and lemon proteomics, considered as recalcitrant tissues in that small amounts of proteins are dispersed into a large body of plant polymers and metabolites. As examples of non-alcoholic beverages, ginger ale, coconut milk, a cola drink, almond milk and orgeat syrup are analyzed. Finally, the trace proteome of white and red wines, beer and aperitifs is reported, with the aim of tracing the industrial manipulations and herbal usage prior to their commercialization. PMID:28234315

Digestion proteomics: tracking lactoferrin truncation and peptide release during simulated gastric digestion.

PubMed

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.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer.

PubMed

Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P

2018-01-01

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

PubMed

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.

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE PAGES

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.; ...

2015-02-16

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

We Are Not Alone: The iMOP Initiative and Its Roles in a Biology- and Disease-Driven Human Proteome Project.

PubMed

Tholey, Andreas; Taylor, Nicolas L; Heazlewood, Joshua L; Bendixen, Emøke

2017-12-01

Mapping of the human proteome has advanced significantly in recent years and will provide a knowledge base to accelerate our understanding of how proteins and protein networks can affect human health and disease. However, providing solutions to human health challenges will likely fail if insights are exclusively based on studies of human samples and human proteomes. In recent years, it has become evident that human health depends on an integrated understanding of the many species that make human life possible. These include the commensal microorganisms that are essential to human life, pathogens, and food species as well as the classic model organisms that enable studies of biological mechanisms. The Human Proteome Organization (HUPO) initiative on multiorganism proteomes (iMOP) works to support proteome research undertaken on nonhuman species that remain widely under-studied compared with the progress in human proteome research. This perspective argues the need for further research on multiple species that impact human life. We also present an update on recent progress in model organisms, microbiota, and food species, address the emerging problem of antibiotics resistance, and outline how iMOP activities could lead to a more inclusive approach for the human proteome project (HPP) to better support proteome research aimed at improving human health and furthering knowledge on human biology.

The State of the Human Proteome in 2013 as viewed through PeptideAtlas: Comparing the Kidney, Urine, and Plasma Proteomes for the Biology and Disease-driven Human Proteome Project

PubMed Central

Farrah, Terry; Deutsch, Eric W.; Omenn, Gilbert S.; Sun, Zhi; Watts, Julian D.; Yamamoto, Tadashi; Shteynberg, David; Harris, Micheleen M.; Moritz, Robert L.

2014-01-01

The kidney, urine, and plasma proteomes are intimately related: proteins and metabolic waste products are filtered from the plasma by the kidney and excreted via the urine, while kidney proteins may be secreted into the circulation or released into the urine. Shotgun proteomics datasets derived from human kidney, urine, and plasma samples were collated and processed using a uniform software pipeline, and relative protein abundances were estimated by spectral counting. The resulting PeptideAtlas builds yielded 4005, 2491, and 3553 nonredundant proteins at 1% FDR for the kidney, urine, and plasma proteomes, respectively—for kidney and plasma, the largest high-confidence protein sets to date. The same pipeline applied to all available human data yielded a 2013 Human PeptideAtlas build containing 12,644 nonredundant proteins and at least one peptide for each of ~14,000 Swiss-Prot entries, an increase over 2012 of ~7.5% of the predicted human proteome. We demonstrate that abundances are correlated between plasma and urine, examine the most abundant urine proteins not derived from either plasma or kidney, and consider the biomarker potential of proteins associated with renal decline. This analysis forms part of the Biology and Disease-driven Human Proteome Project (B/D-HPP) and a contribution to the Chromosome-centric Human Proteome Project (C-HPP) special issue. PMID:24261998

Genome-wide proteomics analysis on longissimus muscles in Qinchuan beef cattle.

PubMed

He, Hua; Chen, Si; Liang, Wei; Liu, Xiaolin

2017-04-01

To gain further insight into the molecular mechanism of bovine muscle development, we combined mass spectrometry characterization of proteins with Illumina deep sequencing of RNAs obtained from bovine longissimus muscle (LD) at prenatal and postnatal stages. For the proteomic study, each group of LD proteins was extracted and labeled using isobaric tags for relative and absolute quantitation (iTRAQ) method. Among the 1321 proteins identified from six samples, 390 proteins were differentially expressed in embryos at day 135 post-fertilization (Emb135d) vs. 30-month-old adult cattle (Emb135d vs. 30M) samples. Gene Ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted to better understand the different functions. Furthermore, we analyzed the relationship between transcript and protein regulation between samples by direct comparison of expression levels from transcriptomic and iTRAQ-based proteomics. Association results indicated that 1295 of 1321 proteins could be mapped to transcriptome sequencing data. This study provides the most comprehensive, targeted survey of bovine LD proteins to date and has shown the power of combining transcriptomic and proteomic approaches to provide molecular insights for understanding the developmental characteristics in bovine muscle, and even in other mammals. © 2016 Stichting International Foundation for Animal Genetics.

Comparative evaluation of saliva collection methods for proteome analysis.

PubMed

Golatowski, Claas; Salazar, Manuela Gesell; Dhople, Vishnu Mukund; Hammer, Elke; Kocher, Thomas; Jehmlich, Nico; Völker, Uwe

2013-04-18

Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses. From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively. Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 μg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed. Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.

PubMed

Wilson, Kate E; Marouga, Rita; Prime, John E; Pashby, D Paul; Orange, Paul R; Crosier, Steven; Keith, Alexander B; Lathe, Richard; Mullins, John; Estibeiro, Peter; Bergling, Helene; Hawkins, Edward; Morris, Christopher M

2005-10-01

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle.

PubMed

Farhoud, Murtada H; Wessels, Hans J C T; Wevers, Ron A; van Engelen, Baziel G; van den Heuvel, Lambert P; Smeitink, Jan A

2005-01-01

In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

PubMed

Inserra, Ilaria; Martelli, Claudia; Cipollina, Mara; Cicione, Claudia; Iavarone, Federica; Taranto, Giuseppe Di; Barba, Marta; Castagnola, Massimo; Desiderio, Claudia; Lattanzi, Wanda

2016-04-01

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins β4 and β10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serum Proteome Signature of Radiation Response: Upregulation of Inflammation-Related Factors and Downregulation of Apolipoproteins and Coagulation Factors in Cancer Patients Treated With Radiation Therapy—A Pilot Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Widlak, Piotr, E-mail: widlak@io.gliwice.pl; Jelonek, Karol; Wojakowska, Anna

Purpose: Ionizing radiation affects the proteome of irradiated cells and tissue, yet data concerning changes induced during radiation therapy (RT) in human blood are fragmentary and inconclusive. We aimed to identify features of serum proteome and associated processes involved in response to partial body irradiation during cancer treatment. Methods and Materials: Twenty patients with head and neck squamous cell cancer (HNSCC) and 20 patients with prostate cancer received definitive intensity modulated RT. Blood samples were collected before RT, just after RT, and 1 month after the end of RT. Complete serum proteome was analyzed in individual samples, using a shotgun liquidmore » chromatography-tandem mass spectrometry approach which allowed identification of approximately 450 proteins. Approximately 100 unique proteins were quantified in all samples after exclusion of immunoglobulins, and statistical significance of differences among consecutive samples was assessed. Processes associated with quantified proteins and their functional interactions were predicted using gene ontology tools. Results: RT-induced changes were marked in the HNSCC patient group: 22 upregulated and 33 downregulated proteins were detected in post-RT sera. Most of the changes reversed during follow-up, yet levels of some proteins remained affected 1 month after the end of RT. RT-upregulated proteins were associated with acute phase, inflammatory response, and complement activation. RT-downregulated proteins were associated with transport and metabolism of lipids (plasma apolipoproteins) and blood coagulation. RT-induced changes were much weaker in prostate cancer patients, which corresponded to differences in acute radiation toxicity observed in both groups. Nevertheless, general patterns of RT-induced sera proteome changes were similar in both of the groups of cancer patients. Conclusions: In this pilot study, we proposed to identify a molecular signature of radiation response, based on specific features of serum proteome. The signature included upregulation of factors involved in acute or inflammatory response but also downregulation of plasma apolipoproteins and factors involved in blood coagulation.« less

MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.

2016-05-03

ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. Themetabolite,protein, andlipidextraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of thismore » protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental,in vitro, and clinical). IMPORTANCEIn systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample.« less

Open Source Software Tool Skyline Reaches Key Agreement with Mass Spectrometer Vendors | Office of Cancer Clinical Proteomics Research

Cancer.gov

The full proteomics analysis of a small tumor sample (similar in mass to a few grains of rice) produces well over 500 megabytes of unprocessed "raw" data when analyzed on a mass spectrometer (MS). Thus, for every proteomics experiment there is a vast amount of raw data that must be analyzed and interrogated in order to extract biological information. Moreover, the raw data output from different MS vendors are generally in different formats inhibiting the ability of labs to productively work together.

Proteomic analysis of formalin-fixed paraffin embedded tissue by MALDI imaging mass spectrometry

PubMed Central

Casadonte, Rita; Caprioli, Richard M

2012-01-01

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

Nanoscale or single cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) platform as a major advance in overall sensitivity. NanoPOTS dramatically enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive LC-MS, nanoPOTS allows identification of ~1500 to ~3,000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runsmore » algorithm of MaxQuant, >3000 proteins were consistently identified from as few as 10 cells. Furthermore, we demonstrate robust quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

PROTEOMIC ANALYSIS OPTIMIZATION: SELECTIVE PROTEIN SAMPLE ON-COLUMN RETENTION IN REVERSE-PHASE LIQUID CHROMATOGRAPHY

EPA Science Inventory

Why work was done?

To be able to identify, on a proteomic level, cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGT) in mouse liver microsomes for the conazole exposure study IRP # NHEERL-ECD-SCN-CZ-2002-01-R1_Addendum 1. The new enrichment method was necessary beca...

Computational approaches to protein inference in shotgun proteomics

PubMed Central

2012-01-01

Shotgun proteomics has recently emerged as a powerful approach to characterizing proteomes in biological samples. Its overall objective is to identify the form and quantity of each protein in a high-throughput manner by coupling liquid chromatography with tandem mass spectrometry. As a consequence of its high throughput nature, shotgun proteomics faces challenges with respect to the analysis and interpretation of experimental data. Among such challenges, the identification of proteins present in a sample has been recognized as an important computational task. This task generally consists of (1) assigning experimental tandem mass spectra to peptides derived from a protein database, and (2) mapping assigned peptides to proteins and quantifying the confidence of identified proteins. Protein identification is fundamentally a statistical inference problem with a number of methods proposed to address its challenges. In this review we categorize current approaches into rule-based, combinatorial optimization and probabilistic inference techniques, and present them using integer programing and Bayesian inference frameworks. We also discuss the main challenges of protein identification and propose potential solutions with the goal of spurring innovative research in this area. PMID:23176300

Proteomic analysis of ligamentum flavum from patients with lumbar spinal stenosis.

PubMed

Kamita, Masahiro; Mori, Taiki; Sakai, Yoshihito; Ito, Sadayuki; Gomi, Masahiro; Miyamoto, Yuko; Harada, Atsushi; Niida, Shumpei; Yamada, Tesshi; Watanabe, Ken; Ono, Masaya

2015-05-01

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64 781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Beyond the proteome: Mass Spectrometry Special Interest Group (MS-SIG) at ISMB/ECCB 2013

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Soyoung; Payne, Samuel H.; Schaab, Christoph

2014-07-02

Mass spectrometry special interest group (MS-SIG) aims to bring together experts from the global research community to discuss highlights and challenges in the field of mass spectrometry (MS)-based proteomics and computational biology. The rapid echnological developments in MS-based proteomics have enabled the generation of a large amount of meaningful information on hundreds to thousands of proteins simultaneously from a biological sample; however, the complexity of the MS data require sophisticated computational algorithms and software for data analysis and interpretation. This year’s MS-SIG meeting theme was ‘Beyond the Proteome’ with major focuses on improving protein identification/quantification and using proteomics data tomore » solve interesting problems in systems biology and clinical research.« less

Making a protein extract from plant pathogenic fungi for gel- and LC-based proteomics.

PubMed

Fernández, Raquel González; Redondo, Inmaculada; Jorrin-Novo, Jesus V

2014-01-01

Proteomic technologies have become a successful tool to provide relevant information on fungal biology. In the case of plant pathogenic fungi, this approach would allow a deeper knowledge of the interaction and the biological cycle of the pathogen, as well as the identification of pathogenicity and virulence factors. These two elements open up new possibilities for crop disease diagnosis and environment-friendly crop protection. Phytopathogenic fungi, due to its particular cellular characteristics, can be considered as a recalcitrant biological material, which makes it difficult to obtain quality protein samples for proteomic analysis. This chapter focuses on protein extraction for gel- and LC-based proteomics with specific protocols of our current research with Botrytis cinerea.

An analysis of the impact of pre-analytical factors on the urine proteome: Sample processing time, temperature, and proteolysis.

PubMed

Hepburn, Sophie; Cairns, David A; Jackson, David; Craven, Rachel A; Riley, Beverley; Hutchinson, Michelle; Wood, Steven; Smith, Matthew Welberry; Thompson, Douglas; Banks, Rosamonde E

2015-06-01

We have examined the impact of sample processing time delay, temperature, and the addition of protease inhibitors (PIs) on the urinary proteome and peptidome, an important aspect of biomarker studies. Ten urine samples from patients with varying pathologies were each divided and PIs added to one-half, with aliquots of each then processed and frozen immediately, or after a delay of 6 h at 4°C or room temperature (20-22°C), effectively yielding 60 samples in total. Samples were then analyzed by 2D-PAGE, SELDI-TOF-MS, and immunoassay. Interindividual variability in profiles was the dominant feature in all analyses. Minimal changes were observed by 2D-PAGE as a result of delay in processing, temperature, or PIs and no changes were seen in IgG, albumin, β2 -microglobulin, or α1 -microglobulin measured by immunoassay. Analysis of peptides showed clustering of some samples by presence/absence of PIs but the extent was very patient-dependent with most samples showing minimal effects. The extent of processing-induced changes and the benefit of PI addition are patient- and sample-dependent. A consistent processing methodology is essential within a study to avoid any confounding of the results. © 2014 The Authors PROTEOMICS Clinical Applications Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Structural Proteomics of Herpesviruses

PubMed Central

Leroy, Baptiste; Gillet, Laurent; Vanderplasschen, Alain; Wattiez, Ruddy

2016-01-01

Herpesviruses are highly prevalent viruses associated with numerous pathologies both in animal and human populations. Until now, most of the strategies used to prevent or to cure these infections have been unsuccessful because these viruses have developed numerous immune evasion mechanisms. Therefore, a better understanding of their complex lifecycle is needed. In particular, while the genome of numerous herpesviruses has been sequenced, the exact composition of virions remains unknown for most of them. Mass spectrometry has recently emerged as a central method and has permitted fundamental discoveries in virology. Here, we review mass spectrometry-based approaches that have recently allowed a better understanding of the composition of the herpesvirus virion. In particular, we describe strategies commonly used for proper sample preparation and fractionation to allow protein localization inside the particle but also to avoid contamination by nonstructural proteins. A collection of other important data regarding post-translational modifications or the relative abundance of structural proteins is also described. This review also discusses the poorly studied importance of host proteins in herpesvirus structural proteins and the necessity to develop a quantitative workflow to better understand the dynamics of the structural proteome. In the future, we hope that this collaborative effort will assist in the development of new strategies to fight these infections. PMID:26907323

Strain-resolved microbial community proteomics reveals simultaneous aerobic and anaerobic function during gastrointestinal tract colonization of a preterm infant

DOE PAGES

Brooks, Brandon; Mueller, R. S.; Young, Jacque C.; ...

2015-07-01

While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13 21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains ofmore » Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.« less

Proteomic analysis of human aqueous humor using multidimensional protein identification technology

PubMed Central

Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

2009-01-01

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

Proteomic Analysis of Serum from Patients with Major Depressive Disorder to Compare Their Depressive and Remission Statuses

PubMed Central

Lee, Jiyeong; Joo, Eun-Jeong; Lim, Hee-Joung; Park, Jong-Moon; Lee, Kyu Young; Park, Arum; Seok, AeEun

2015-01-01

Objective Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. Methods Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. Results The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. Conclusion This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation. PMID:25866527

Liquid Chromatography Mass Spectrometry-Based Proteomics: Biological and Technological Aspects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpievitch, Yuliya V.; Polpitiya, Ashoka D.; Anderson, Gordon A.

2010-12-01

Mass spectrometry-based proteomics has become the tool of choice for identifying and quantifying the proteome of an organism. Though recent years have seen a tremendous improvement in instrument performance and the computational tools used, significant challenges remain, and there are many opportunities for statisticians to make important contributions. In the most widely used "bottom-up" approach to proteomics, complex mixtures of proteins are first subjected to enzymatic cleavage, the resulting peptide products are separated based on chemical or physical properties and analyzed using a mass spectrometer. The two fundamental challenges in the analysis of bottom-up MS-based proteomics are: (1) Identifying themore » proteins that are present in a sample, and (2) Quantifying the abundance levels of the identified proteins. Both of these challenges require knowledge of the biological and technological context that gives rise to observed data, as well as the application of sound statistical principles for estimation and inference. We present an overview of bottom-up proteomics and outline the key statistical issues that arise in protein identification and quantification.« less

Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges.

PubMed

Maryáš, Josef; Faktor, Jakub; Dvořáková, Monika; Struhárová, Iva; Grell, Peter; Bouchal, Pavel

2014-03-01

Metastases are responsible for most of the cases of death in patients with solid tumors. There is thus an urgent clinical need of better understanding the exact molecular mechanisms and finding novel therapeutics targets and biomarkers of metastatic disease of various tumors. Metastases are formed in a complicated biological process called metastatic cascade. Up to now, proteomics has enabled the identification of number of metastasis-associated proteins and potential biomarkers in cancer tissues, microdissected cells, model systems, and secretomes. Expression profiles and biological role of key proteins were confirmed in verification and functional experiments. This communication reviews these observations and analyses the methodological aspects of the proteomics approaches used. Moreover, it reviews contribution of current proteomics in the field of functional characterization and interactome analysis of proteins involved in various events in metastatic cascade. It is evident that ongoing technical progress will further increase proteome coverage and sample capacity of proteomics technologies, giving complex answers to clinical and functional questions asked. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A complete mass spectrometric map for the analysis of the yeast proteome and its application to quantitative trait analysis

PubMed Central

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

Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

PubMed Central

Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2017-01-01

In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

PubMed Central

Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S; Ellisman, Mark H; Ting, Alice Y

2018-01-01

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). PMID:28796234

Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

PubMed

Säll, Anna; Persson, Helena; Ohlin, Mats; Borrebaeck, Carl A K; Wingren, Christer

2016-09-25

Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Statistical Design for Biospecimen Cohort Size in Proteomics-based Biomarker Discovery and Verification Studies

PubMed Central

Skates, Steven J.; Gillette, Michael A.; LaBaer, Joshua; Carr, Steven A.; Anderson, N. Leigh; Liebler, Daniel C.; Ransohoff, David; Rifai, Nader; Kondratovich, Marina; Težak, Živana; Mansfield, Elizabeth; Oberg, Ann L.; Wright, Ian; Barnes, Grady; Gail, Mitchell; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Boja, Emily S.

2014-01-01

Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC), with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance, and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step towards building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research. PMID:24063748

Statistical design for biospecimen cohort size in proteomics-based biomarker discovery and verification studies.

PubMed

Skates, Steven J; Gillette, Michael A; LaBaer, Joshua; Carr, Steven A; Anderson, Leigh; Liebler, Daniel C; Ransohoff, David; Rifai, Nader; Kondratovich, Marina; Težak, Živana; Mansfield, Elizabeth; Oberg, Ann L; Wright, Ian; Barnes, Grady; Gail, Mitchell; Mesri, Mehdi; Kinsinger, Christopher R; Rodriguez, Henry; Boja, Emily S

2013-12-06

Protein biomarkers are needed to deepen our understanding of cancer biology and to improve our ability to diagnose, monitor, and treat cancers. Important analytical and clinical hurdles must be overcome to allow the most promising protein biomarker candidates to advance into clinical validation studies. Although contemporary proteomics technologies support the measurement of large numbers of proteins in individual clinical specimens, sample throughput remains comparatively low. This problem is amplified in typical clinical proteomics research studies, which routinely suffer from a lack of proper experimental design, resulting in analysis of too few biospecimens to achieve adequate statistical power at each stage of a biomarker pipeline. To address this critical shortcoming, a joint workshop was held by the National Cancer Institute (NCI), National Heart, Lung, and Blood Institute (NHLBI), and American Association for Clinical Chemistry (AACC) with participation from the U.S. Food and Drug Administration (FDA). An important output from the workshop was a statistical framework for the design of biomarker discovery and verification studies. Herein, we describe the use of quantitative clinical judgments to set statistical criteria for clinical relevance and the development of an approach to calculate biospecimen sample size for proteomic studies in discovery and verification stages prior to clinical validation stage. This represents a first step toward building a consensus on quantitative criteria for statistical design of proteomics biomarker discovery and verification research.

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study.

PubMed

Lee, Hye Min; Gupta, Ravi; Kim, Sun Hyung; Wang, Yiming; Rakwal, Randeep; Agrawal, Ganesh Kumar; Kim, Sun Tae

2015-05-01

High-abundance proteins (HAPs) hamper in-depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose-dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM-prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low-abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang-geum and Ho-bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI-TOF-TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

PubMed

Jiang, Xiao-Sheng; Dai, Jie; Sheng, Quan-Hu; Zhang, Lei; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong

2005-01-01

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

Proteomic Profiling of Serial Prediagnostic Serum Samples for Early Detection of Colon Cancer in the U.S. Military.

PubMed

Shao, Stephanie; Neely, Benjamin A; Kao, Tzu-Cheg; Eckhaus, Janet; Bourgeois, Jolie; Brooks, Jasmin; Jones, Elizabeth E; Drake, Richard R; Zhu, Kangmin

2017-05-01

Background: Serum proteomic biomarkers offer a promising approach for early detection of cancer. In this study, we aimed to identify proteomic profiles that could distinguish colon cancer cases from controls using serial prediagnostic serum samples. Methods: This was a nested case-control study of active duty military members. Cases consisted of 264 patients diagnosed with colon cancer between 2001 and 2009. Controls were matched to cases on age, gender, race, serum sample count, and collection date. We identified peaks that discriminated cases from controls using random forest data analysis with a 2/3 training and 1/3 validation dataset. We then included epidemiologic data to see whether further improvement of model performance was obtainable. Proteins that corresponded to discriminatory peaks were identified. Results: Peaks with m/z values of 3,119.32, 2,886.67, 2,939.23, and 5,078.81 were found to discriminate cases from controls with a sensitivity of 69% and a specificity of 67% in the year before diagnosis. When smoking status was included, sensitivity increased to 76% while histories of other cancer and tonsillectomy raised specificity to 76%. Peaks at 2,886.67 and 3,119.32 m/z were identified as histone acetyltransferases while 2,939.24 m/z was a transporting ATPase subunit. Conclusions: Proteomic profiles in the year before cancer diagnosis have the potential to discriminate colon cancer patients from controls, and the addition of epidemiologic information may increase the sensitivity and specificity of discrimination. Impact: Our findings indicate the potential value of using serum prediagnostic proteomic biomarkers in combination with epidemiologic data for early detection of colon cancer. Cancer Epidemiol Biomarkers Prev; 26(5); 711-8. ©2016 AACR . ©2016 American Association for Cancer Research.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.

PubMed

Jiang, Xiaogang; Feng, Shun; Tian, Ruijun; Han, Guanghui; Jiang, Xinning; Ye, Mingliang; Zou, Hanfa

2007-02-01

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.

Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

PubMed

Laganowsky, Arthur; Gómez, Stephen M; Whitelegge, Julian P; Nishio, John N

2009-04-13

The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

Transitioning from Targeted to Comprehensive Mass Spectrometry Using Genetic Algorithms.

PubMed

Jaffe, Jacob D; Feeney, Caitlin M; Patel, Jinal; Lu, Xiaodong; Mani, D R

2016-11-01

Targeted proteomic assays are becoming increasingly popular because of their robust quantitative applications enabled by internal standardization, and they can be routinely executed on high performance mass spectrometry instrumentation. However, these assays are typically limited to 100s of analytes per experiment. Considerable time and effort are often expended in obtaining and preparing samples prior to targeted analyses. It would be highly desirable to detect and quantify 1000s of analytes in such samples using comprehensive mass spectrometry techniques (e.g., SWATH and DIA) while retaining a high degree of quantitative rigor for analytes with matched internal standards. Experimentally, it is facile to port a targeted assay to a comprehensive data acquisition technique. However, data analysis challenges arise from this strategy concerning agreement of results from the targeted and comprehensive approaches. Here, we present the use of genetic algorithms to overcome these challenges in order to configure hybrid targeted/comprehensive MS assays. The genetic algorithms are used to select precursor-to-fragment transitions that maximize the agreement in quantification between the targeted and the comprehensive methods. We find that the algorithm we used provided across-the-board improvement in the quantitative agreement between the targeted assay data and the hybrid comprehensive/targeted assay that we developed, as measured by parameters of linear models fitted to the results. We also found that the algorithm could perform at least as well as an independently-trained mass spectrometrist in accomplishing this task. We hope that this approach will be a useful tool in the development of quantitative approaches for comprehensive proteomics techniques. Graphical Abstract ᅟ.

An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.

2010-02-05

A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL.more » In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking level present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects, but in turn limits the achievable dynamic range compared to the TOF detector.« less

Transitioning from Targeted to Comprehensive Mass Spectrometry Using Genetic Algorithms

NASA Astrophysics Data System (ADS)

Jaffe, Jacob D.; Feeney, Caitlin M.; Patel, Jinal; Lu, Xiaodong; Mani, D. R.

2016-11-01

Targeted proteomic assays are becoming increasingly popular because of their robust quantitative applications enabled by internal standardization, and they can be routinely executed on high performance mass spectrometry instrumentation. However, these assays are typically limited to 100s of analytes per experiment. Considerable time and effort are often expended in obtaining and preparing samples prior to targeted analyses. It would be highly desirable to detect and quantify 1000s of analytes in such samples using comprehensive mass spectrometry techniques (e.g., SWATH and DIA) while retaining a high degree of quantitative rigor for analytes with matched internal standards. Experimentally, it is facile to port a targeted assay to a comprehensive data acquisition technique. However, data analysis challenges arise from this strategy concerning agreement of results from the targeted and comprehensive approaches. Here, we present the use of genetic algorithms to overcome these challenges in order to configure hybrid targeted/comprehensive MS assays. The genetic algorithms are used to select precursor-to-fragment transitions that maximize the agreement in quantification between the targeted and the comprehensive methods. We find that the algorithm we used provided across-the-board improvement in the quantitative agreement between the targeted assay data and the hybrid comprehensive/targeted assay that we developed, as measured by parameters of linear models fitted to the results. We also found that the algorithm could perform at least as well as an independently-trained mass spectrometrist in accomplishing this task. We hope that this approach will be a useful tool in the development of quantitative approaches for comprehensive proteomics techniques.

Enhancing Bottom-up and Top-down Proteomic Measurements with Ion Mobility Separations

DOE PAGES

Baker, Erin Shammel; Burnum-Johnson, Kristin E.; Ibrahim, Yehia M.; ...

2015-07-03

Proteomic measurements with greater throughput, sensitivity and additional structural information enhance the in-depth characterization of complex mixtures and targeted studies with additional information and higher confidence. While liquid chromatography separation coupled with mass spectrometry (LC-MS) measurements have provided information on thousands of proteins in different sample types, the additional of another rapid separation stage providing structural information has many benefits for analyses. Technical advances in ion funnels and multiplexing have enabled ion mobility separations to be easily and effectively coupled with LC-MS proteomics to enhance the information content of measurements. Finally, herein, we report on applications illustrating increased sensitivity, throughput,more » and structural information by utilizing IMS-MS and LC-IMS-MS measurements for both bottom-up and top-down proteomics measurements.« less

Recent advances on multidimensional liquid chromatography-mass spectrometry for proteomics: from qualitative to quantitative analysis--a review.

PubMed

Wu, Qi; Yuan, Huiming; Zhang, Lihua; Zhang, Yukui

2012-06-20

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography-mass spectrometry (MDLC-MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including "top-down" and "bottom-up" to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

PubMed

Bourgeois, Michael; Jacquin, Françoise; Savois, Vincent; Sommerer, Nicolas; Labas, Valérie; Henry, Céline; Burstin, Judith

2009-01-01

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post-translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

[Proteomics in infectious diseases].

PubMed

Quero, Sara; Párraga-Niño, Noemí; García-Núñez, Marian; Sabrià, Miquel

2016-04-01

Infectious diseases have a high incidence in the population, causing a major impact on global health. In vitro culture of microorganisms is the first technique applied for infection diagnosis which is laborious and time consuming. In recent decades, efforts have been focused on the applicability of "Omics" sciences, highlighting the progress provided by proteomic techniques in the field of infectious diseases. This review describes the management, processing and analysis of biological samples for proteomic research. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

High-throughput sperm differential proteomics suggests that epigenetic alterations contribute to failed assisted reproduction.

PubMed

Azpiazu, Rubén; Amaral, Alexandra; Castillo, Judit; Estanyol, Josep Maria; Guimerà, Marta; Ballescà, Josep Lluís; Balasch, Juan; Oliva, Rafael

2014-06-01

Are there quantitative alterations in the proteome of normozoospermic sperm samples that are able to complete IVF but whose female partner does not achieve pregnancy? Normozoospermic sperm samples with different IVF outcomes (pregnancy versus no pregnancy) differed in the levels of at least 66 proteins. The analysis of the proteome of sperm samples with distinct fertilization capacity using low-throughput proteomic techniques resulted in the detection of a few differential proteins. Current high-throughput mass spectrometry approaches allow the identification and quantification of a substantially higher number of proteins. This was a case-control study including 31 men with normozoospermic sperm and their partners who underwent IVF with successful fertilization recruited between 2007 and 2008. Normozoospermic sperm samples from 15 men whose female partners did not achieve pregnancy after IVF (no pregnancy) and 16 men from couples that did achieve pregnancy after IVF (pregnancy) were included in this study. To perform the differential proteomic experiments, 10 no pregnancy samples and 10 pregnancy samples were separately pooled and subsequently used for tandem mass tags (TMT) protein labelling, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry (LC-MS/MS) identification and peak intensity relative protein quantification. Bioinformatic analyses were performed using UniProt Knowledgebase, DAVID and Reactome. Individual samples (n = 5 no pregnancy samples; n = 6 pregnancy samples) and aliquots from the above TMT pools were used for western blotting. By using TMT labelling and LC-MS/MS, we have detected 31 proteins present at lower abundance (ratio no pregnancy/pregnancy < 0.67) and 35 at higher abundance (ratio no pregnancy/pregnancy > 1.5) in the no pregnancy group. Bioinformatic analyses showed that the proteins with differing abundance are involved in chromatin assembly and lipoprotein metabolism (P values < 0.05). In addition, the differential abundance of one of the proteins (SRSF protein kinase 1) was further validated by western blotting using independent samples (P value < 0.01). For individual samples the amount of recovered sperm not used for IVF was low and in most of the cases insufficient for MS analysis, therefore pools of samples had to be used to this end. Alterations in the proteins involved in chromatin assembly and metabolism may result in epigenetic errors during spermatogenesis, leading to inaccurate sperm epigenetic signatures, which could ultimately prevent embryonic development. These sperm proteins may thus possibly have clinical relevance. This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economia y Competividad; FEDER BFU 2009-07118 and PI13/00699) and Fundación Salud 2000 SERONO13-015. There are no competing interests to declare.

Proteomic analysis of human dental cementum and alveolar bone

PubMed Central

Salmon, Cristiane R.; Tomazela, Daniela M.; Ruiz, Karina Gonzales Silvério; Foster, Brian L.; Leme, Adriana Franco Paes; Sallum, Enilson Antonio; Somerman, Martha J.; Nociti, Francisco H.

2013-01-01

Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration. PMID:24007660

Analyzing large-scale proteomics projects with latent semantic indexing.

PubMed

Klie, Sebastian; Martens, Lennart; Vizcaíno, Juan Antonio; Côté, Richard; Jones, Phil; Apweiler, Rolf; Hinneburg, Alexander; Hermjakob, Henning

2008-01-01

Since the advent of public data repositories for proteomics data, readily accessible results from high-throughput experiments have been accumulating steadily. Several large-scale projects in particular have contributed substantially to the amount of identifications available to the community. Despite the considerable body of information amassed, very few successful analyses have been performed and published on this data, leveling off the ultimate value of these projects far below their potential. A prominent reason published proteomics data is seldom reanalyzed lies in the heterogeneous nature of the original sample collection and the subsequent data recording and processing. To illustrate that at least part of this heterogeneity can be compensated for, we here apply a latent semantic analysis to the data contributed by the Human Proteome Organization's Plasma Proteome Project (HUPO PPP). Interestingly, despite the broad spectrum of instruments and methodologies applied in the HUPO PPP, our analysis reveals several obvious patterns that can be used to formulate concrete recommendations for optimizing proteomics project planning as well as the choice of technologies used in future experiments. It is clear from these results that the analysis of large bodies of publicly available proteomics data by noise-tolerant algorithms such as the latent semantic analysis holds great promise and is currently underexploited.

Proteogenomics Dashboard for the Human Proteome Project.

PubMed

Tabas-Madrid, Daniel; Alves-Cruzeiro, Joao; Segura, Victor; Guruceaga, Elizabeth; Vialas, Vital; Prieto, Gorka; García, Carlos; Corrales, Fernando J; Albar, Juan Pablo; Pascual-Montano, Alberto

2015-09-04

dasHPPboard is a novel proteomics-based dashboard that collects and reports the experiments produced by the Spanish Human Proteome Project consortium (SpHPP) and aims to help HPP to map the entire human proteome. We have followed the strategy of analog genomics projects like the Encyclopedia of DNA Elements (ENCODE), which provides a vast amount of data on human cell lines experiments. The dashboard includes results of shotgun and selected reaction monitoring proteomics experiments, post-translational modifications information, as well as proteogenomics studies. We have also processed the transcriptomics data from the ENCODE and Human Body Map (HBM) projects for the identification of specific gene expression patterns in different cell lines and tissues, taking special interest in those genes having little proteomic evidence available (missing proteins). Peptide databases have been built using single nucleotide variants and novel junctions derived from RNA-Seq data that can be used in search engines for sample-specific protein identifications on the same cell lines or tissues. The dasHPPboard has been designed as a tool that can be used to share and visualize a combination of proteomic and transcriptomic data, providing at the same time easy access to resources for proteogenomics analyses. The dasHPPboard can be freely accessed at: http://sphppdashboard.cnb.csic.es.

Candidate-based proteomics in the search for biomarkers of cardiovascular disease

PubMed Central

Anderson, Leigh

2005-01-01

The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a ‘directed proteomics’ approach. PMID:15611012

Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).

PubMed

Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd

2011-01-01

The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.

A novel one-class SVM based negative data sampling method for reconstructing proteome-wide HTLV-human protein interaction networks.

PubMed

Mei, Suyu; Zhu, Hao

2015-01-26

Protein-protein interaction (PPI) prediction is generally treated as a problem of binary classification wherein negative data sampling is still an open problem to be addressed. The commonly used random sampling is prone to yield less representative negative data with considerable false negatives. Meanwhile rational constraints are seldom exerted on model selection to reduce the risk of false positive predictions for most of the existing computational methods. In this work, we propose a novel negative data sampling method based on one-class SVM (support vector machine, SVM) to predict proteome-wide protein interactions between HTLV retrovirus and Homo sapiens, wherein one-class SVM is used to choose reliable and representative negative data, and two-class SVM is used to yield proteome-wide outcomes as predictive feedback for rational model selection. Computational results suggest that one-class SVM is more suited to be used as negative data sampling method than two-class PPI predictor, and the predictive feedback constrained model selection helps to yield a rational predictive model that reduces the risk of false positive predictions. Some predictions have been validated by the recent literature. Lastly, gene ontology based clustering of the predicted PPI networks is conducted to provide valuable cues for the pathogenesis of HTLV retrovirus.

Proteome Comparisons between Hemolymph of Two Honeybee Strains (Apis mellifera ligustica) Reveal Divergent Molecular Basis in Driving Hemolymph Function and High Royal Jelly Secretion.

PubMed

Ararso, Zewdu; Ma, Chuan; Qi, Yuping; Feng, Mao; Han, Bin; Hu, Han; Meng, Lifeng; Li, Jianke

2018-01-05

Hemolymph is vital for the immunity of honeybees and offers a way to investigate their physiological status. To gain novel insight into the functionality and molecular details of the hemolymph in driving increased Royal Jelly (RJ) production, we characterized and compared hemolymph proteomes across the larval and adult ages of Italian bees (ITbs) and Royal Jelly bees (RJbs), a stock selected from ITbs for increasing RJ output. Unprecedented in-depth proteome was attained with the identification of 3394 hemolymph proteins in both bee lines. The changes in proteome support the general function of hemolymph to drive development and immunity across different ages. However, age-specific proteome settings have adapted to prime the distinct physiology for larvae and adult bees. In larvae, the proteome is thought to drive temporal immunity, rapid organogenesis, and reorganization of larval structures. In adults, the proteome plays key roles in prompting tissue development and immune defense in newly emerged bees, in gland maturity in nurse bees, and in carbohydrate energy production in forager bees. Between larval and adult samples of the same age, RJbs and ITbs have tailored distinct hemolymph proteome programs to drive their physiology. In particular, in day 4 larvae and nurse bees, a large number of highly abundant proteins are enriched in protein synthesis and energy metabolism in RJbs. This implies that they have adapted their proteome to initiate different developmental trajectories and high RJ secretion in response to selection for enhanced RJ production. Our hitherto unexplored in-depth proteome coverage provides novel insight into molecular details that drive hemolymph function and high RJ production by RJbs.

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

PubMed Central

Trevisan-Silva, Dilza; Bednaski, Aline V.; Fischer, Juliana S.G.; Veiga, Silvio S.; Bandeira, Nuno; Guthals, Adrian; Marchini, Fabricio K.; Leprevost, Felipe V.; Barbosa, Valmir C.; Senff-Ribeiro, Andrea; Carvalho, Paulo C.

2017-01-01

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523. PMID:28696408

Efficient visualization of high-throughput targeted proteomics experiments: TAPIR.

PubMed

Röst, Hannes L; Rosenberger, George; Aebersold, Ruedi; Malmström, Lars

2015-07-15

Targeted mass spectrometry comprises a set of powerful methods to obtain accurate and consistent protein quantification in complex samples. To fully exploit these techniques, a cross-platform and open-source software stack based on standardized data exchange formats is required. We present TAPIR, a fast and efficient Python visualization software for chromatograms and peaks identified in targeted proteomics experiments. The input formats are open, community-driven standardized data formats (mzML for raw data storage and TraML encoding the hierarchical relationships between transitions, peptides and proteins). TAPIR is scalable to proteome-wide targeted proteomics studies (as enabled by SWATH-MS), allowing researchers to visualize high-throughput datasets. The framework integrates well with existing automated analysis pipelines and can be extended beyond targeted proteomics to other types of analyses. TAPIR is available for all computing platforms under the 3-clause BSD license at https://github.com/msproteomicstools/msproteomicstools. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Polymethacrylate-based monoliths as stationary phases for separation of biopolymers and immobilization of enzymes.

PubMed

Martinović, Tamara; Josić, Djuro

2017-11-01

The experiences in the production and application of polymethacrylate-based monolithic supports, since their development almost thirty years ago, are presented. The main driving force for the development of new chromatographic supports was the necessity for the isolation and separation of physiologically active biopolymers and their use for therapeutic purposes. For this sake, a development of a method for fast separation, preventing denaturation and preserving their biological activity was necessary. Development of polysaccharide-based supports, followed by the introduction of polymer-based chromatographic media, is shortly described. This development was followed by the advances in monolithic media that are now used for both large- and small-scale separation of biopolymers and nanoparticles. Finally, a short overview is given about the applications of monoliths for sample displacement chromatography, resulting in isolation of physiologically active biomolecules, such as proteins, protein complexes, and nucleic acid, as well as high-throughput sample preparation for proteomic investigations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A chronic fatigue syndrome – related proteome in human cerebrospinal fluid

PubMed Central

Baraniuk, James N; Casado, Begona; Maibach, Hilda; Clauw, Daniel J; Pannell, Lewis K; Hess S, Sonja

2005-01-01

Background Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects. Methods Cerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 μl/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 μl/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis. Results Pooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of ≥1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were α-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described. Conclusion This pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared. PMID:16321154

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

PubMed

Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

2016-04-01

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. Copyright © 2016 Elsevier B.V. All rights reserved.

Simplifying the human serum proteome for discriminating patients with bipolar disorder of other psychiatry conditions.

PubMed

de Jesus, Jemmyson Romário; Galazzi, Rodrigo Moretto; de Lima, Tatiani Brenelli; Banzato, Cláudio Eduardo Muller; de Almeida Lima E Silva, Luiz Fernando; de Rosalmeida Dantas, Clarissa; Gozzo, Fábio Cézar; Arruda, Marco Aurélio Zezzi

2017-12-01

An exploratory analysis using proteomic strategies in blood serum of patients with bipolar disorder (BD), and with other psychiatric conditions such as Schizophrenia (SCZ), can provide a better understanding of this disorder, as well as their discrimination based on their proteomic profile. The proteomic profile of blood serum samples obtained from patients with BD using lithium or other drugs (N=14), healthy controls, including non-family (HCNF; N=3) and family (HCF; N=9), patients with schizophrenia (SCZ; N=23), and patients using lithium for other psychiatric conditions (OD; N=4) were compared. Four methods for simplifying the serum samples proteome were evaluated for both removing the most abundant proteins and for enriching those of lower-abundance: protein depletion with acetonitrile (ACN), dithiothreitol (DTT), sequential depletion using DTT and ACN, and protein equalization using commercial ProteoMiner® kit (PM). For proteomic evaluation, 2-D DIGE and nanoLC-MS/MS analysis were employed. PM method was the best strategy for removing proteins of high abundance. Through 2-D DIGE gel image comparison, 37 protein spots were found differentially abundant (p<0.05, Student's t-test), which exhibited ≥2.0-fold change of the average value of normalized spot intensities in the serum of SCZ, BD and OD patients compared to subject controls (HCF and HCNF). From these spots detected, 13 different proteins were identified: ApoA1, ApoE, ApoC3, ApoA4, Samp, SerpinA1, TTR, IgK, Alb, VTN, TR, C4A and C4B. Proteomic analysis allowed the discrimination of patients with BD from patients with other mental disorders, such as SCZ. The findings in this exploratory study may also contribute for better understanding the pathophysiology of these disorders and finding potential serum biomarkers for these conditions. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Altered proteomic polymorphisms in the caterpillar body and stroma of natural Cordyceps sinensis during maturation.

PubMed

Dong, Yun-Zi; Zhang, Li-Juan; Wu, Zi-Mei; Gao, Ling; Yao, Yi-Sang; Tan, Ning-Zhi; Wu, Jian-Yong; Ni, Luqun; Zhu, Jia-Shi

2014-01-01

To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%-4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs.

Altered Proteomic Polymorphisms in the Caterpillar Body and Stroma of Natural Cordyceps sinensis during Maturation

PubMed Central

Wu, Zi-Mei; Gao, Ling; Yao, Yi-Sang; Tan, Ning-Zhi; Wu, Jian-Yong; Ni, Luqun; Zhu, Jia-Shi

2014-01-01

Objective To examine the maturational changes in proteomic polymorphisms resulting from differential expression by multiple intrinsic fungi in the caterpillar body and stroma of natural Cordyceps sinensis (Cs), an integrated micro-ecosystem. Methods The surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) biochip technique was used to profile the altered protein compositions in the caterpillar body and stroma of Cs during its maturation. The MS chromatograms were analyzed using density-weighted algorithms to examine the similarities and cluster relationships among the proteomic polymorphisms of the Cs compartments and the mycelial products Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph). Results: SELDI-TOF MS chromatograms displayed dynamic proteomic polymorphism alterations among samples from the different Cs compartments during maturation. More than 1,900 protein bands were analyzed using density-weighted ZUNIX similarity equations and clustering methods, revealing integral polymorphism similarities of 57.4% between the premature and mature stromata and 42.8% between the premature and mature caterpillar bodies. The across-compartment similarity was low, ranging from 10.0% to 18.4%. Consequently, each Cs compartment (i.e., the stroma and caterpillar body) formed a clustering clade, and the 2 clades formed a Cs cluster. The polymorphic similarities ranged from 0.51% to 1.04% between Hs and the Cs compartments and were 2.8- to 4.8-fold higher (1.92%–4.34%) between Ph and the Cs compartments. The Hs and Ph mycelial samples formed isolated clades outside of the Cs cluster. Conclusion Proteomic polymorphisms in the caterpillar body and stroma of Cs change dynamically during maturation. The proteomic polymorphisms in Hs and Ph differ from those in Cs, suggesting the presence of multiple Cs-associated fungi and multiple Ophiocordyceps sinensis genotypes with altered differential protein expression in the Cs compartments during maturation. In conjunction with prior mycological and molecular observations, the findings from this proteomic study support the integrated micro-ecosystem hypothesis for natural Cs. PMID:25310818

Early Prediction of Lupus Nephritis Using Advanced Proteomics

DTIC Science & Technology

2012-06-01

urine samples for research were obtained, and information on the following laboratory measures was collected: BUN ( urea ), serum creatinine, serum... urine chemistry), medications and other clinical outcomes (overall disease activity, renal and overall damage). Specific Aim 2: Advanced proteomic...measured by the external standards. We concluded that serial measurements of plasma and urine NGAL may be valuable in predicting impending worsening of

NOVEL CONTINUOUS PH/SALT GRADIENT AND PEPTIDE SCORE FOR STRONG CATION EXCHANGE CHROMATOGRAPHY IN 2D-NANO-LC/MSMS PEPTIDE IDENTIFICATION FOR PROTEOMICS

EPA Science Inventory

Tryptic digests of human serum albumin (HSA) and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MSMS with strong cation exchange (SCX) and reverse phase (RP) chromatography and contin...

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes.

PubMed

Wang, Guanghui; Wu, Wells W; Zeng, Weihua; Chou, Chung-Lin; Shen, Rong-Fong

2006-05-01

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.

Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simpson, David C.; Smith, Richard D.

2005-04-01

Throughout the field of global proteomics, ranging from simple organism studies to human medical applications, the high sample complexity creates demands for improved separations and analysis techniques. Furthermore, with increased organism complexity, the correlation between proteome and genome becomes less certain due to extensive mRNA processing prior to translation. In this way, the same DNA sequence can potentially code for regions in a number of distinct proteins; quantitative differences in expression (or abundance) between these often-related species are of significant interest. Well-established proteomics techniques, which use genomic information to identify peptides that originate from protease digestion, often cannot easily distinguishmore » between such gene products; intact protein-level analyses are required to complete the picture, particularly for identifying post-translational modifications. While chromatographic techniques are currently better suited to peptide analysis, capillary electrophoresis (CE) in combination with mass spectrometry (MS) may become important for intact protein analysis. This review focuses on CE/MS instrumentation and techniques showing promise for such applications, highlighting those with greatest potential. Reference will also be made to developments relevant to peptide-level analyses for use in time- or sample-limited situations.« less

A tutorial in displaying mass spectrometry-based proteomic data using heat maps.

PubMed

Key, Melissa

2012-01-01

Data visualization plays a critical role in interpreting experimental results of proteomic experiments. Heat maps are particularly useful for this task, as they allow us to find quantitative patterns across proteins and biological samples simultaneously. The quality of a heat map can be vastly improved by understanding the options available to display and organize the data in the heat map. This tutorial illustrates how to optimize heat maps for proteomics data by incorporating known characteristics of the data into the image. First, the concepts used to guide the creating of heat maps are demonstrated. Then, these concepts are applied to two types of analysis: visualizing spectral features across biological samples, and presenting the results of tests of statistical significance. For all examples we provide details of computer code in the open-source statistical programming language R, which can be used for biologists and clinicians with little statistical background. Heat maps are a useful tool for presenting quantitative proteomic data organized in a matrix format. Understanding and optimizing the parameters used to create the heat map can vastly improve both the appearance and the interoperation of heat map data.

Effects of exercise on the urinary proteome.

PubMed

Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

2015-01-01

Exercise-induced proteinuria has been observed and studied for more than a century. It was found that different sport disciplines alter the urinary proteome in different ways. Moderate-intensity exercise results in increased glomerular filtration, meaning that medium-sized proteins are excreted in higher amounts, while high-intensity exercise of short duration also increases the excretion of low molecular weight proteins as a result of tubular dysfunction. Exhaustive exercise may lead to the excretion of hemoglobin or myoglobin, which changes the urinary proteome considerably. Studies comparing protein maps of different sport types compared to a control group showed that quality and quantity of urinary proteins are interindividually different. In addition, urine samples collected before and after exercise exhibit substantially different protein patterns even from the same person. Therefore, further studies investigating the urinary proteome are desirable. As the variation of protein content and composition in urine are generally much higher than in other matrices, respective studies need to be well controlled and homogenous groups of volunteers should be chosen. In addition to the sport-related physiological and biochemical interest, exercise-induced protein changes also need to be considered for biomarker measurements from urine samples for kidney or other diseases.

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry*

PubMed Central

Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.

2016-01-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. PMID:27670688

Informed-Proteomics: open-source software package for top-down proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jungkap; Piehowski, Paul D.; Wilkins, Christopher

Top-down proteomics involves the analysis of intact proteins. This approach is very attractive as it allows for analyzing proteins in their endogenous form without proteolysis, preserving valuable information about post-translation modifications, isoforms, proteolytic processing or their combinations collectively called proteoforms. Moreover, the quality of the top-down LC-MS/MS datasets is rapidly increasing due to advances in the liquid chromatography and mass spectrometry instrumentation and sample processing protocols. However, the top-down mass spectra are substantially more complex compare to the more conventional bottom-up data. To take full advantage of the increasing quality of the top-down LC-MS/MS datasets there is an urgent needmore » to develop algorithms and software tools for confident proteoform identification and quantification. In this study we present a new open source software suite for top-down proteomics analysis consisting of an LC-MS feature finding algorithm, a database search algorithm, and an interactive results viewer. The presented tool along with several other popular tools were evaluated using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.« less

Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases.

PubMed

Cho, Christine H; Nuttall, Mark E

2002-12-01

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.

The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.

PubMed

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.

Low-Molecular-Weight Plasma Proteome Analysis Using Top-Down Mass Spectrometry.

PubMed

Cheon, Dong Huey; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

2017-01-01

While human plasma has a wealth of diagnostic information regarding the state of the human body in heath and disease, low molecular weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we describe a protocol for top-down proteomic analysis to identify and characterize the LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. Each type of plasma sample was first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC software. As a result, a total of 442 LMW proteins and cleaved products, including those with posttranslational modifications (PTMs) and single amino acid variations (SAAVs), were identified with a threshold E-value of 1 × 10 -4 from the four types of plasma samples.

A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

2013-02-01

Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred frommore » one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.« less

Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

PubMed

Sato, Nori; Taniguchi, Takako; Goda, Yuichiro; Kosaka, Hirofumi; Higashino, Kosaku; Sakai, Toshinori; Katoh, Shinsuke; Yasui, Natsuo; Sairyo, Koichi; Taniguchi, Hisaaki

2016-12-02

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Establishment of the optimum two-dimensional electrophoresis system of ovine ovarian tissue.

PubMed

Jia, J L; Zhang, L P; Wu, J P; Wang, J; Ding, Q

2014-08-26

Lambing performance of sheep is the most important economic trait and is regarded as a critic factoring affecting the productivity in sheep industry. Ovary plays the most roles in lambing trait. To establish the optimum two-dimensional electrophoresis system (2-DE) of ovine ovarian tissue, the common protein extraction methods of animal tissue (trichloroacetic acid/acetone precipitation and direct schizolysis methods) were used to extract ovine ovarian protein, and 17-cm nonlinear immobilized PH 3-10 gradient strips were used for 2-DE. The sample handling, loading quantity of the protein sample, and isoelectric focusing (IEF) steps were manipulated and optimized in this study. The results indicate that the direct schizolysis III method, a 200-μg loading quantity of the protein sample, and IEF steps II (20°C active hydration, 14 h→500 V, 1 h→1000 V 1 h→1000-9000 V, 6 h→80,000 VH→500 V 24 h) are optimal for 2-DE analysis of ovine ovarian tissue. Therefore, ovine ovarian tissue proteomics 2-DE was preliminarily established by the optimized conditions in this study; meanwhile, the conditions identified herein could provide a reference for ovarian sample preparation and 2-DE using tissues from other animals.

Identification of IGFBP2 and IGFBP3 As Compensatory Biomarkers for CA19-9 in Early-Stage Pancreatic Cancer Using a Combination of Antibody-Based and LC-MS/MS-Based Proteomics

PubMed Central

Yoneyama, Toshihiro; Ohtsuki, Sumio; Honda, Kazufumi; Kobayashi, Makoto; Iwasaki, Motoki; Uchida, Yasuo; Okusaka, Takuji; Nakamori, Shoji; Shimahara, Masashi; Ueno, Takaaki; Tsuchida, Akihiko; Sata, Naohiro; Ioka, Tatsuya; Yasunami, Yohichi; Kosuge, Tomoo; Kaneda, Takashi; Kato, Takao; Yagihara, Kazuhiro; Fujita, Shigeyuki; Huang, Wilber; Yamada, Tesshi; Tachikawa, Masanori; Terasaki, Tetsuya

2016-01-01

Pancreatic cancer is one of the most lethal tumors, and reliable detection of early-stage pancreatic cancer and risk diseases for pancreatic cancer is essential to improve the prognosis. As 260 genes were previously reported to be upregulated in invasive ductal adenocarcinoma of pancreas (IDACP) cells, quantification of the corresponding proteins in plasma might be useful for IDACP diagnosis. Therefore, the purpose of the present study was to identify plasma biomarkers for early detection of IDACP by using two proteomics strategies: antibody-based proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Among the 260 genes, we focused on 130 encoded proteins with known function for which antibodies were available. Twenty-three proteins showed values of the area under the curve (AUC) of more than 0.8 in receiver operating characteristic (ROC) analysis of reverse-phase protein array (RPPA) data of IDACP patients compared with healthy controls, and these proteins were selected as biomarker candidates. We then used our high-throughput selected reaction monitoring or multiple reaction monitoring (SRM/MRM) methodology, together with an automated sample preparation system, micro LC and auto analysis system, to quantify these candidate proteins in plasma from healthy controls and IDACP patients on a large scale. The results revealed that insulin-like growth factor-binding protein (IGFBP)2 and IGFBP3 have the ability to discriminate IDACP patients at an early stage from healthy controls, and IGFBP2 appeared to be increased in risk diseases of pancreatic malignancy, such as intraductal papillary mucinous neoplasms (IPMNs). Furthermore, diagnosis of IDACP using the combination of carbohydrate antigen 19–9 (CA19-9), IGFBP2 and IGFBP3 is significantly more effective than CA19-9 alone. This suggests that IGFBP2 and IGFBP3 may serve as compensatory biomarkers for CA19-9. Early diagnosis with this marker combination may improve the prognosis of IDACP patients. PMID:27579675

Progress and pitfalls in finding the 'missing proteins' from the human proteome map.

PubMed

Segura, Victor; Garin-Muga, Alba; Guruceaga, Elizabeth; Corrales, Fernando J

2017-01-01

The Human Proteome Project was launched with two main goals: the comprehensive and systematic definition of the human proteome map and the development of ready to use analytical tools to measure relevant proteins in their biological context in health and disease. Despite the great progress in this endeavour, there is still a group of reluctant proteins with no, or scarce, experimental evidence supporting their existence. These are called the 'missing proteins' and represent one of the biggest challenges to complete the human proteome map. Areas covered: This review focuses on the description of the missing proteome based on the HUPO standards, the analysis of the reasons explaining the difficulty of detecting missing proteins and the strategies currently used in the search for missing proteins. The present and future of the quest for the missing proteins is critically revised hereafter. Expert commentary: An overarching multidisciplinary effort is currently being done under the HUPO umbrella to allow completion of the human proteome map. It is expected that the detection of missing proteins will grow in the coming years since the methods and the best tissue/cell type sample for their search are already on the table.

UNiquant, a program for quantitative proteomics analysis using stable isotope labeling.

PubMed

Huang, Xin; Tolmachev, Aleksey V; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A; Smith, Richard D; Chan, Wing C; Hinrichs, Steven H; Fu, Kai; Ding, Shi-Jian

2011-03-04

Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for postmeasurement normalization of peptide ratios, which is required by the other programs.

UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

PubMed Central

Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven H.; Fu, Kai; Ding, Shi-Jian

2011-01-01

Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs. PMID:21158445

Proteomic analyses of host and pathogen responses during bovine mastitis.

PubMed

Boehmer, Jamie L

2011-12-01

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced.

Chip-LC-MS for label-free profiling of human serum.

PubMed

Horvatovich, Peter; Govorukhina, Natalia I; Reijmers, Theo H; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer

2007-12-01

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics.

PubMed

Poersch, Aline; Grassi, Mariana Lopes; Carvalho, Vinícius Pereira de; Lanfredi, Guilherme Pauperio; Palma, Camila de Souza; Greene, Lewis Joel; de Sousa, Christiani Bisinoto; Carrara, Hélio Humberto Angotti; Candido Dos Reis, Francisco José; Faça, Vitor Marcel

2016-08-11

Tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in the context of proteomics studies. To uncover differences among benign and malignant ovarian samples, we performed a quantitative proteomic study consisting of albumin immunodepletion, isotope labeling with acrylamide and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histological type. 1135 proteins were identified, corresponding to 505 gene products. 223 proteins presented associated quantification and the comparative analysis of histological types revealed 75 differentially abundant proteins. Based on this, we developed a panel for targeted proteomic analysis using the multiple reaction monitoring (MRM) method for validation of 51 proteins in individual samples of high-grade serous ovarian tumor fluids (malignant) and benign serous cystadenoma tumor fluids. This analysis showed concordant results in terms of average amounts of proteins, and APOE, SERPINF2, SERPING1, ADAM17, CD44 and OVGP1 were statistically significant between benign and malignant group. The results observed in the MRM for APOE were confirmed by western blotting, where APOE was more abundant in malignant samples. This molecular signature can contribute to improve tumor stratification and shall be investigated in combination with current biomarkers in larger cohorts to improve ovarian cancer diagnosis. Despite advances in cancer research, ovarian cancer has a high mortality and remains a major challenge due to a number of particularities of the disease, especially late diagnosis caused by vague clinical symptoms, the cellular and molecular heterogeneity of tumors, and the lack of effective treatment. Thus, efforts are directed to better understand this neoplasia, its origin, development and, particularly the identification and validation of biomarkers for early detection of the disease in asymptomatic stage. In the present work, we confirmed by MRM method in individual ovarian tumor fluid samples the regulation of 27 proteins out of 33 identified in a highthroughput study. We speculate that the presence and/or differential abundance observed in tumor fluid is a cooperation primarily of high rates of secretion of such tumor proteins to extra tumor environment that will at the end accumulate in plasma, and also the accumulation of acute-phase proteins throughout the entire body. On top of that, consideration of physiological influences in the interpretation of expression observed, including age, menopause status, route-of-elimination kinetics and metabolism of the tumor marker, coexisting disease, hormonal imbalances, life-style influences (smoking, alcoholism, obesity), among others, are mandatory to enable the selection of good protein tumor marker candidates for extensive validation. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

A flexible statistical model for alignment of label-free proteomics data – incorporating ion mobility and product ion information

PubMed Central

2013-01-01

Background The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing - the matching of peptide measurements across samples. Results We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Conclusions Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods. PMID:24341404

A flexible statistical model for alignment of label-free proteomics data--incorporating ion mobility and product ion information.

PubMed

Benjamin, Ashlee M; Thompson, J Will; Soderblom, Erik J; Geromanos, Scott J; Henao, Ricardo; Kraus, Virginia B; Moseley, M Arthur; Lucas, Joseph E

2013-12-16

The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing--the matching of peptide measurements across samples. We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods.

Reproducible Tissue Homogenization and Protein Extraction for Quantitative Proteomics Using MicroPestle-Assisted Pressure-Cycling Technology.

PubMed

Shao, Shiying; Guo, Tiannan; Gross, Vera; Lazarev, Alexander; Koh, Ching Chiek; Gillessen, Silke; Joerger, Markus; Jochum, Wolfram; Aebersold, Ruedi

2016-06-03

The reproducible and efficient extraction of proteins from biopsy samples for quantitative analysis is a critical step in biomarker and translational research. Recently, we described a method consisting of pressure-cycling technology (PCT) and sequential windowed acquisition of all theoretical fragment ions-mass spectrometry (SWATH-MS) for the rapid quantification of thousands of proteins from biopsy-size tissue samples. As an improvement of the method, we have incorporated the PCT-MicroPestle into the PCT-SWATH workflow. The PCT-MicroPestle is a novel, miniaturized, disposable mechanical tissue homogenizer that fits directly into the microTube sample container. We optimized the pressure-cycling conditions for tissue lysis with the PCT-MicroPestle and benchmarked the performance of the system against the conventional PCT-MicroCap method using mouse liver, heart, brain, and human kidney tissues as test samples. The data indicate that the digestion of the PCT-MicroPestle-extracted proteins yielded 20-40% more MS-ready peptide mass from all tissues tested with a comparable reproducibility when compared to the conventional PCT method. Subsequent SWATH-MS analysis identified a higher number of biologically informative proteins from a given sample. In conclusion, we have developed a new device that can be seamlessly integrated into the PCT-SWATH workflow, leading to increased sample throughput and improved reproducibility at both the protein extraction and proteomic analysis levels when applied to the quantitative proteomic analysis of biopsy-level samples.

Application of proteomics in the discovery of candidate protein biomarkers in a Diabetes Autoantibody Standardization Program (DASP) sample subset

PubMed Central

Metz, Thomas O.; Qian, Wei-Jun; Jacobs, Jon M.; Gritsenko, Marina A.; Moore, Ronald J.; Polpitiya, Ashoka D.; Monroe, Matthew E.; Camp, David G.; Mueller, Patricia W.; Smith, Richard D.

2009-01-01

Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted “-omics” approaches are under-utilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. Alpha-2-glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples. PMID:18092746

Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset.

PubMed

Metz, Thomas O; Qian, Wei-Jun; Jacobs, Jon M; Gritsenko, Marina A; Moore, Ronald J; Polpitiya, Ashoka D; Monroe, Matthew E; Camp, David G; Mueller, Patricia W; Smith, Richard D

2008-02-01

Novel biomarkers of type 1 diabetes must be identified and validated in initial, exploratory studies before they can be assessed in proficiency evaluations. Currently, untargeted "-omics" approaches are underutilized in profiling studies of clinical samples. This report describes the evaluation of capillary liquid chromatography (LC) coupled with mass spectrometry (MS) in a pilot proteomic analysis of human plasma and serum from a subset of control and type 1 diabetic individuals enrolled in the Diabetes Autoantibody Standardization Program, with the goal of identifying candidate biomarkers of type 1 diabetes. Initial high-resolution capillary LC-MS/MS experiments were performed to augment an existing plasma peptide database, while subsequent LC-FTICR studies identified quantitative differences in the abundance of plasma proteins. Analysis of LC-FTICR proteomic data identified five candidate protein biomarkers of type 1 diabetes. alpha-2-Glycoprotein 1 (zinc), corticosteroid-binding globulin, and lumican were 2-fold up-regulated in type 1 diabetic samples relative to control samples, whereas clusterin and serotransferrin were 2-fold up-regulated in control samples relative to type 1 diabetic samples. Observed perturbations in the levels of all five proteins are consistent with the metabolic aberrations found in type 1 diabetes. While the discovery of these candidate protein biomarkers of type 1 diabetes is encouraging, follow up studies are required for validation in a larger population of individuals and for determination of laboratory-defined sensitivity and specificity values using blinded samples.

In-Source Fragmentation and the Sources of Partially Tryptic Peptides in Shotgun Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong-Seo; Monroe, Matthew E.; Camp, David G.

2013-02-01

Partially tryptic peptides are often identified in shotgun proteomics using trypsin as the proteolytic enzyme; however, it has been controversial regarding the sources of such partially tryptic peptides. Herein we investigate the impact of in-source fragmentation on shotgun proteomics using three biological samples, including a standard protein mixture, a mouse brain tissue homogenate, and a mouse plasma sample. Since the in-source fragments of a peptide retain the same elution time with its parent fully tryptic peptide, the partially tryptic peptides from in-source fragmentation can be distinguished from the other partially tryptic peptides by plotting the observed retention times against themore » computationally predicted retention times. Most partially tryptic in-source fragmentation artifacts were misaligned from the linear distribution of fully tryptic peptides. The impact of in-source fragmentation on peptide identifications was clearly significant in a less complex sample such as a standard protein digest, where ~60 % of unique peptides were observed as partially tryptic peptides from in-source fragmentation. In mouse brain or mouse plasma samples, in-source fragmentation contributed to 1-3 % of all identified peptides. The other major source of partially tryptic peptides in complex biological samples is presumably proteolytic processing by endogenous proteases in the samples. By filtering out the in-source fragmentation artifacts from the identified partially tryptic or non-tryptic peptides, it is possible to directly survey in-vivo proteolytic processing in biological samples such as blood plasma.« less

Purification and proteomic analysis of plant plasma membranes.

PubMed

Alexandersson, Erik; Gustavsson, Niklas; Bernfur, Katja; Karlsson, Adine; Kjellbom, Per; Larsson, Christer

2008-01-01

All techniques needed for proteomic analyses of plant plasma membranes are described in detail, from isolation of plasma membranes to protein identification by mass spectrometry (MS). Plasma membranes are isolated by aqueous two-phase partitioning yielding vesicles with a cytoplasmic side-in orientation and a purity of about 95%. These vesicles are turned inside-out by treatment with Brij 58, which removes soluble contaminating proteins enclosed in the vesicles as well as loosely attached proteins. The final plasma membrane preparation thus retains all integral proteins and many peripheral proteins. Proteins are separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands are excised and digested with trypsin. Peptides in tryptic digests are separated by nanoflow liquid chromatography and either fed directly into an ESI-MS or spotted onto matrix-assisted laser desorption ionization (MALDI) plates for analysis with MALDI-MS. Finally, data processing and database searching are used for protein identification to define a plasma membrane proteome.

Limited proteolysis in proteomics using protease-immobilized microreactors.

PubMed

Yamaguchi, Hiroshi; Miyazaki, Masaya; Maeda, Hideaki

2012-01-01

Proteolysis is the key step for proteomic studies integrated with MS analysis. Compared with the conventional method of in-solution digestion, proteolysis by a protease-immobilized microreactor has a number of advantages for proteomic analysis; i.e., rapid and efficient digestion, elimination of a purification step of the digests prior to MS, and high stability against a chemical or thermal denaturant. This chapter describes the preparation of the protease-immobilized microreactors and proteolysis performance of these microreactors. Immobilization of proteases by the formation of a polymeric membrane consisting solely of protease-proteins on the inner wall of the microchannel is performed. This was realized either by a cross-linking reaction in a laminar flow between lysine residues sufficiently present on the protein surfaces themselves or in the case of acidic proteins by mixing them with poly-lysine prior to the crosslink-reaction. The present procedure is simple and widely useful not only for proteases but also for several other enzymes.

MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses

PubMed Central

Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C.; Burnum-Johnson, Kristin E.; Kim, Young-Mo; Kyle, Jennifer E.; Matzke, Melissa M.; Shukla, Anil K.; Chu, Rosalie K.; Schepmoes, Athena A.; Jacobs, Jon M.; Baric, Ralph S.; Webb-Robertson, Bobbie-Jo; Smith, Richard D.

2016-01-01

ABSTRACT Integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. Despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. Here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. The metabolite, protein, and lipid extraction (MPLEx) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. To illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with Middle East respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. The MPLEx method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). IMPORTANCE In systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. Thus, the prospect of extracting different types of molecules (e.g., DNAs, RNAs, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. Here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. Author Video: An author video summary of this article is available. PMID:27822525

Comparative proteomic analysis reveals that T3SS, Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri.

PubMed

Facincani, Agda P; Moreira, Leandro M; Soares, Márcia R; Ferreira, Cristiano B; Ferreira, Rafael M; Ferro, Maria I T; Ferro, Jesus A; Gozzo, Fabio C; de Oliveira, Julio C F

2014-03-01

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant-pathogen interactions.

Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection.

PubMed

Andringa, Kelly K; King, Adrienne L; Eccleston, Heather B; Mantena, Sudheer K; Landar, Aimee; Jhala, Nirag C; Dickinson, Dale A; Squadrito, Giuseppe L; Bailey, Shannon M

2010-05-01

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

Proteomics with Mass Spectrometry Imaging: Beyond Amyloid Typing.

PubMed

Lavatelli, Francesca; Merlini, Giampaolo

2018-04-01

Detection and typing of amyloid deposits in tissues are two crucial steps in the management of systemic amyloidoses. The presence of amyloid deposits is routinely evaluated through Congo red staining, whereas proteomics is now a mainstay in the identification of the deposited proteins. In article number 1700236, Winter et al. [Proteomics 2017, 17, Issue 22] describe a novel method based on MALDI-MS imaging coupled to ion mobility separation and peptide filtering, to detect the presence of amyloid in histology samples and to identify its composition, while preserving the spatial distribution of proteins in tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advances in microscale separations towards nanoproteomics applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Lian; Piehowski, Paul D.; Shi, Tujin

Microscale separations (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. While significant advances have been achieved in MS-based proteomics, the current platforms still face a significant challenge in overall sensitivity towards nanoproteomics (i.e., with less than 1 g total amount of proteins available) applications such as cellular heterogeneity in tissue pathologies. Herein, we review recent advances in microscale separation techniques and integrated sample processing systems that improve the overall sensitivity and coverage of the proteomics workflow, and their contributionsmore » towards nanoproteomics applications.« less

Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

ERIC Educational Resources Information Center

Kim, Thomas D.; Craig, Paul A.

2010-01-01

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

Proteome-wide analysis of Anopheles culicifacies mosquito midgut: new insights into the mechanism of refractoriness.

PubMed

Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Singh, Jagbir; Adak, Tridibesh; Sharma, Arun

2018-05-08

Midgut invasion, a major bottleneck for malaria parasites transmission is considered as a potential target for vector-parasite interaction studies. New intervention strategies are required to explore the midgut proteins and their potential role in refractoriness for malaria control in Anopheles mosquitoes. To better understand the midgut functional proteins of An. culicifacies susceptible and refractory species, proteomic approaches coupled with bioinformatics analysis is an effective means in order to understand the mechanism of refractoriness. In the present study, an integrated in solution- in gel trypsin digestion approach, along with Isobaric tag for relative and absolute quantitation (iTRAQ)-Liquid chromatography/Mass spectrometry (LC/MS/MS) and data mining were performed to identify the proteomic profile and differentially expressed proteins in Anopheles culicifacies susceptible species A and refractory species B. Shot gun proteomics approaches led to the identification of 80 proteins in An. culicifacies susceptible species A and 92 in refractory species B and catalogue was prepared. iTRAQ based proteomic analysis identified 48 differentially expressed proteins from total 130 proteins. Of these, 41 were downregulated and 7 were upregulated in refractory species B in comparison to susceptible species A. We report that the altered midgut proteins identified in naturally refractory mosquitoes are involved in oxidative phosphorylation, antioxidant and proteolysis process that may suggest their role in parasite growth inhibition. Furthermore, real time polymerase chain reaction (PCR) analysis of few proteins indicated higher expression of iTRAQ upregulated protein in refractory species than susceptible species. This study elucidates the first proteome of the midguts of An. culicifacies sibling species that attempts to analyze unique proteogenomic interactions to provide insights for better understanding of the mechanism of refractoriness. Functional implications of these upregulated proteins in refractory species may reflect the phenotypic characteristics of the mosquitoes and will improve our understandings of blood meal digestion process, parasite vector interactions and proteomes of other vectors of human diseases for development of novel vector control strategies.

Proteome profile and biological activity of caprine, bovine and human milk fat globules.

PubMed

Spertino, Stefano; Cipriani, Valentina; De Angelis, Chiara; Giuffrida, Maria Gabriella; Marsano, Francesco; Cavaletto, Maria

2012-04-01

Upon combining bidimensional electrophoresis with monodimensional separation, a more comprehensive analysis of the milk fat globule membrane has been obtained. The proteomic profile of caprine milk fat globules revealed the presence of butyrophilin, lactadherin and perilipin as the major proteins, they were also associated to bovine and human milk fat globule membranes. Xanthine dehydrogenase/oxidase has been detected only in monodimensional gels. Biological activity of milk fat globules has been evaluated in Caco2-cells, as a representative model of the intestinal barrier. The increase of cell viability was indicative of a potential nutraceutical role for the whole milk fat globule, suggesting a possible employment in milk formula preparation.

Effects of Three Commonly-used Diuretics on the Urinary Proteome

PubMed Central

Li, Xundou; Zhao, Mindi; Li, Menglin; Jia, Lulu; Gao, Youhe

2014-01-01

Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F; hydrochlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS). Based on the criteria of P ⩽ 0.05, a fold change ⩾2, a spectral count ⩾5, and false positive rate (FDR) ⩽1%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC–MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics. PMID:24508280

Characterization of the human aqueous humour proteome: A comparison of the genders.

PubMed

Perumal, Natarajan; Manicam, Caroline; Steinicke, Matthias; Funke, Sebastian; Pfeiffer, Norbert; Grus, Franz H

2017-01-01

Aqueous humour (AH) is an important biologic fluid that maintains normal intraocular pressure and contains proteins that regulate the homeostasis of ocular tissues. Any alterations in the protein compositions are correlated to the pathogenesis of various ocular disorders. In recent years, gender-based medicine has emerged as an important research focus considering the prevalence of certain diseases, which are higher in a particular sex. Nevertheless, the inter-gender variations in the AH proteome are unknown. Therefore, this study endeavoured to characterize the AH proteome to assess the differences between genders. Thirty AH samples of patients who underwent cataract surgery were categorized according to their gender. Label-free quantitative discovery mass spectrometry-based proteomics strategy was employed to characterize the AH proteome. A total of 147 proteins were identified with a false discovery rate of less than 1% and only the top 10 major AH proteins make up almost 90% of the total identified proteins. A large number of proteins identified were correlated to defence, immune and inflammatory mechanisms, and response to wounding. Four proteins were found to be differentially abundant between the genders, comprising SERPINF1, SERPINA3, SERPING1 and PTGDS. The findings emerging from our study provide the first insight into the gender-based proteome differences in the AH and also highlight the importance in considering potential sex-dependent changes in the proteome of ocular pathologies in future studies employing the AH.

Consolidation of proteomics data in the Cancer Proteomics database.

PubMed

Arntzen, Magnus Ø; Boddie, Paul; Frick, Rahel; Koehler, Christian J; Thiede, Bernd

2015-11-01

Cancer is a class of diseases characterized by abnormal cell growth and one of the major reasons for human deaths. Proteins are involved in the molecular mechanisms leading to cancer, furthermore they are affected by anti-cancer drugs, and protein biomarkers can be used to diagnose certain cancer types. Therefore, it is important to explore the proteomics background of cancer. In this report, we developed the Cancer Proteomics database to re-interrogate published proteome studies investigating cancer. The database is divided in three sections related to cancer processes, cancer types, and anti-cancer drugs. Currently, the Cancer Proteomics database contains 9778 entries of 4118 proteins extracted from 143 scientific articles covering all three sections: cell death (cancer process), prostate cancer (cancer type) and platinum-based anti-cancer drugs including carboplatin, cisplatin, and oxaliplatin (anti-cancer drugs). The detailed information extracted from the literature includes basic information about the articles (e.g., PubMed ID, authors, journal name, publication year), information about the samples (type, study/reference, prognosis factor), and the proteomics workflow (Subcellular fractionation, protein, and peptide separation, mass spectrometry, quantification). Useful annotations such as hyperlinks to UniProt and PubMed were included. In addition, many filtering options were established as well as export functions. The database is freely available at http://cancerproteomics.uio.no. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioinformatics strategies in life sciences: from data processing and data warehousing to biological knowledge extraction.

PubMed

Thiele, Herbert; Glandorf, Jörg; Hufnagel, Peter

2010-05-27

With the large variety of Proteomics workflows, as well as the large variety of instruments and data-analysis software available, researchers today face major challenges validating and comparing their Proteomics data. Here we present a new generation of the ProteinScape bioinformatics platform, now enabling researchers to manage Proteomics data from the generation and data warehousing to a central data repository with a strong focus on the improved accuracy, reproducibility and comparability demanded by many researchers in the field. It addresses scientists; current needs in proteomics identification, quantification and validation. But producing large protein lists is not the end point in Proteomics, where one ultimately aims to answer specific questions about the biological condition or disease model of the analyzed sample. In this context, a new tool has been developed at the Spanish Centro Nacional de Biotecnologia Proteomics Facility termed PIKE (Protein information and Knowledge Extractor) that allows researchers to control, filter and access specific information from genomics and proteomic databases, to understand the role and relationships of the proteins identified in the experiments. Additionally, an EU funded project, ProDac, has coordinated systematic data collection in public standards-compliant repositories like PRIDE. This will cover all aspects from generating MS data in the laboratory, assembling the whole annotation information and storing it together with identifications in a standardised format.

Substrate-Mediated Laser Ablation under Ambient Conditions for Spatially-Resolved Tissue Proteomics

PubMed Central

Fatou, Benoit; Wisztorski, Maxence; Focsa, Cristian; Salzet, Michel; Ziskind, Michael; Fournier, Isabelle

2015-01-01

Numerous applications of ambient Mass Spectrometry (MS) have been demonstrated over the past decade. They promoted the emergence of various micro-sampling techniques such as Laser Ablation/Droplet Capture (LADC). LADC consists in the ablation of analytes from a surface and their subsequent capture in a solvent droplet which can then be analyzed by MS. LADC is thus generally performed in the UV or IR range, using a wavelength at which analytes or the matrix absorb. In this work, we explore the potential of visible range LADC (532 nm) as a micro-sampling technology for large-scale proteomics analyses. We demonstrate that biomolecule analyses using 532 nm LADC are possible, despite the low absorbance of biomolecules at this wavelength. This is due to the preponderance of an indirect substrate-mediated ablation mechanism at low laser energy which contrasts with the conventional direct ablation driven by sample absorption. Using our custom LADC system and taking advantage of this substrate-mediated ablation mechanism, we were able to perform large-scale proteomic analyses of micro-sampled tissue sections and demonstrated the possible identification of proteins with relevant biological functions. Consequently, the 532 nm LADC technique offers a new tool for biological and clinical applications. PMID:26674367

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

PubMed

Wu, Pei-Wen; Mason, Katelyn E; Durbin-Johnson, Blythe P; Salemi, Michelle; Phinney, Brett S; Rocke, David M; Parker, Glendon J; Rice, Robert H

2017-07-01

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry.

PubMed

Bailey, Ulla-Maja; Punyadeera, Chamindie; Cooper-White, Justin J; Schulz, Benjamin L

2012-12-12

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. Copyright © 2012 Elsevier B.V. All rights reserved.

Intraluminal proteome and peptidome of human urinary extracellular vesicles.

PubMed

Liu, Xinyu; Chinello, Clizia; Musante, Luca; Cazzaniga, Marta; Tataruch, Dorota; Calzaferri, Giulio; James Smith, Andrew; De Sio, Gabriele; Magni, Fulvio; Zou, Hequn; Holthofer, Harry

2015-06-01

Urinary extracellular vesicles (UEVs) are a novel source for disease biomarker discovery. However, Tamm-Horsfall protein (THP) is still a challenge for proteomic analysis since it can inhibit detection of low-abundance proteins. Here, we introduce a new approach that does not involve an ultracentrifugation step to enrich vesicles and that reduces the amount of THP to manageable levels. UEVs were dialyzed and ultrafiltered after reduction and alkylation. The retained fraction was digested with trypsin to reduce the remaining THP and incubated with deoxycholate (DOC). The internal peptidome and internal proteome were analyzed by LC-ESI-MS. A total of 942 different proteins and 3115 unique endogenous peptide fragments deriving from 973 different protein isoforms were identified. Around 82% of the key endosomal sorting complex required for transport components of UEVs generation could be detected from the intraluminal content. Our UEVs preparation protocol provides a simplified way to investigate the intraluminal proteome and peptidome, in particular the subpopulation of UEVs of the trypsin-resistant class of exosomes (positive for tumor susceptibility gene101) and eliminates the majority of interfering proteins such as THP. This method allows the possibility to study endoproteome and endopeptidome of UEVs, thus greatly facilitating biomarker discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Systematic Characterization of the Murine Mitochondrial Proteome Using Functionally Validated Cardiac Mitochondria

PubMed Central

Zhang, Jun; Li, Xiaohai; Mueller, Michael; Wang, Yueju; Zong, Chenggong; Deng, Ning; Vondriska, Thomas M.; Liem, David A.; Yang, Jeong-In; Korge, Paavo; Honda, Henry; Weiss, James N.; Apweiler, Rolf; Ping, Peipei

2009-01-01

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism and biogenesis. In addition, there are several other clusters--including proteolysis, protein folding, and reduction/oxidation signaling-which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles. PMID:18348319

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

PubMed

Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

2015-03-01

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2015-10-21

Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines

PubMed Central

Chen, Yao-Yi; Dasari, Surendra; Ma, Ze-Qiang; Vega-Montoto, Lorenzo J.; Li, Ming

2013-01-01

Spectral counting has become a widely used approach for measuring and comparing protein abundance in label-free shotgun proteomics. However, when analyzing complex samples, the ambiguity of matching between peptides and proteins greatly affects the assessment of peptide and protein inventories, differentiation, and quantification. Meanwhile, the configuration of database searching algorithms that assign peptides to MS/MS spectra may produce different results in comparative proteomic analysis. Here, we present three strategies to improve comparative proteomics through spectral counting. We show that comparing spectral counts for peptide groups rather than for protein groups forestalls problems introduced by shared peptides. We demonstrate the advantage and flexibility of this new method in two datasets. We present four models to combine four popular search engines that lead to significant gains in spectral counting differentiation. Among these models, we demonstrate a powerful vote counting model that scales well for multiple search engines. We also show that semi-tryptic searching outperforms tryptic searching for comparative proteomics. Overall, these techniques considerably improve protein differentiation on the basis of spectral count tables. PMID:22552787

Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines.

PubMed

Chen, Yao-Yi; Dasari, Surendra; Ma, Ze-Qiang; Vega-Montoto, Lorenzo J; Li, Ming; Tabb, David L

2012-09-01

Spectral counting has become a widely used approach for measuring and comparing protein abundance in label-free shotgun proteomics. However, when analyzing complex samples, the ambiguity of matching between peptides and proteins greatly affects the assessment of peptide and protein inventories, differentiation, and quantification. Meanwhile, the configuration of database searching algorithms that assign peptides to MS/MS spectra may produce different results in comparative proteomic analysis. Here, we present three strategies to improve comparative proteomics through spectral counting. We show that comparing spectral counts for peptide groups rather than for protein groups forestalls problems introduced by shared peptides. We demonstrate the advantage and flexibility of this new method in two datasets. We present four models to combine four popular search engines that lead to significant gains in spectral counting differentiation. Among these models, we demonstrate a powerful vote counting model that scales well for multiple search engines. We also show that semi-tryptic searching outperforms tryptic searching for comparative proteomics. Overall, these techniques considerably improve protein differentiation on the basis of spectral count tables.

Saliva Proteomics Analysis Offers Insights on Type 1 Diabetes Pathology in a Pediatric Population

PubMed Central

Pappa, Eftychia; Vastardis, Heleni; Mermelekas, George; Gerasimidi-Vazeou, Andriani; Zoidakis, Jerome; Vougas, Konstantinos

2018-01-01

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease. PMID:29755368

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

PubMed

Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

2007-03-01

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

The Multinational Arabidopsis Steering Subcommittee for Proteomics Assembles the Largest Proteome Database Resource for Plant Systems Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weckwerth, Wolfram; Baginsky, Sacha; Van Wijk, Klass

2009-12-01

In the past 10 years, we have witnessed remarkable advances in the field of plant molecular biology. The rapid development of proteomic technologies and the speed with which these techniques have been applied to the field have altered our perception of how we can analyze proteins in complex systems. At nearly the same time, the availability of the complete genome for the model plant Arabidopsis thaliana was released; this effort provides an unsurpassed resource for the identification of proteins when researchers use MS to analyze plant samples. Recognizing the growth in this area, the Multinational Arabidopsis Steering Committee (MASC) establishedmore » a subcommittee for A. thaliana proteomics in 2006 with the objective of consolidating databases, technique standards, and experimentally validated candidate genes and functions. Since the establishment of the Multinational Arabidopsis Steering Subcommittee for Proteomics (MASCP), many new approaches and resources have become available. Recently, the subcommittee established a webpage to consolidate this information (www.masc-proteomics.org). It includes links to plant proteomic databases, general information about proteomic techniques, meeting information, a summary of proteomic standards, and other relevant resources. Altogether, this website provides a useful resource for the Arabidopsis proteomics community. In the future, the website will host discussions and investigate the cross-linking of databases. The subcommittee members have extensive experience in arabidopsis proteomics and collectively have produced some of the most extensive proteomics data sets for this model plant (Table S1 in the Supporting Information has a list of resources). The largest collection of proteomics data from a single study in A. thaliana was assembled into an accessible database (AtProteome; http://fgcz-atproteome.unizh.ch/index.php) and was recently published by the Baginsky lab.1 The database provides links to major Arabidopsis online resources, and raw data have been deposited in PRIDE and PRIDE BioMart. Included in this database is an Arabidopsis proteome map that provides evidence for the expression of {approx}50% of all predicted gene models, including several alternative gene models that are not represented in The Arabidopsis Information Resource (TAIR) protein database. A set of organ-specific biomarkers is provided, as well as organ-specific proteotypic peptides for 4105 proteins that can be used to facilitate targeted quantitative proteomic surveys. In the future, the AtProteome database will be linked to additional existing resources developed by MASCP members, such as PPDB, ProMEX, and SUBA. The most comprehensive study on the Arabidopsis chloroplast proteome, which includes information on chloroplast sorting signals, posttranslational modifications (PTMs), and protein abundances (analyzed by high-accuracy MS [Orbitrap]), was recently published by the van Wijk lab.2 These and previous data are available via the plant proteome database (PPDB; http://ppdb.tc.cornell.edu) for A. thaliana and maize. PPDB provides genome-wide experimental and functional characterization of the A. thaliana and maize proteomes, including PTMs and subcellular localization information, with an emphasis on leaf and plastid proteins. Maize and Arabidopsis proteome entries are directly linked via internal BLAST alignments within PPDB. Direct links for each protein to TAIR, SUBA, ProMEX, and other resources are also provided.« less

Dithiothreitol-based protein equalization technology to unravel biomarkers for bladder cancer.

PubMed

Araújo, J E; López-Fernández, H; Diniz, M S; Baltazar, Pedro M; Pinheiro, Luís Campos; da Silva, Fernando Calais; Carrascal, Mylène; Videira, Paula; Santos, H M; Capelo, J L

2018-04-01

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Statistical Analysis of Variation in the Human Plasma Proteome

DOE PAGES

Corzett, Todd H.; Fodor, Imola K.; Choi, Megan W.; ...

2010-01-01

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where onemore » human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.« less

A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus

PubMed Central

Hajduk, Joanna; Klupczynska, Agnieszka; Dereziński, Paweł; Matysiak, Jan; Kokot, Piotr; Nowak, Dorota M.; Gajęcka, Marzena; Nowak-Markwitz, Ewa; Kokot, Zenon J.

2015-01-01

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, l-citrulline, l-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases. PMID:26694367

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

PubMed

Burnum-Johnson, Kristin E; Nie, Song; Casey, Cameron P; Monroe, Matthew E; Orton, Daniel J; Ibrahim, Yehia M; Gritsenko, Marina A; Clauss, Therese R W; Shukla, Anil K; Moore, Ronald J; Purvine, Samuel O; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S; Smith, Richard D

2016-12-01

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Statistical analysis of variation in the human plasma proteome.

PubMed

Corzett, Todd H; Fodor, Imola K; Choi, Megan W; Walsworth, Vicki L; Turteltaub, Kenneth W; McCutchen-Maloney, Sandra L; Chromy, Brett A

2010-01-01

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.

Proteogenomics approaches for studying cancer biology and their potential in the identification of acute myeloid leukemia biomarkers.

PubMed

Hernandez-Valladares, Maria; Vaudel, Marc; Selheim, Frode; Berven, Frode; Bruserud, Øystein

2017-08-01

Mass spectrometry (MS)-based proteomics has become an indispensable tool for the characterization of the proteome and its post-translational modifications (PTM). In addition to standard protein sequence databases, proteogenomics strategies search the spectral data against the theoretical spectra obtained from customized protein sequence databases. Up to date, there are no published proteogenomics studies on acute myeloid leukemia (AML) samples. Areas covered: Proteogenomics involves the understanding of genomic and proteomic data. The intersection of both datatypes requires advanced bioinformatics skills. A standard proteogenomics workflow that could be used for the study of AML samples is described. The generation of customized protein sequence databases as well as bioinformatics tools and pipelines commonly used in proteogenomics are discussed in detail. Expert commentary: Drawing on evidence from recent cancer proteogenomics studies and taking into account the public availability of AML genomic data, the interpretation of present and future MS-based AML proteomic data using AML-specific protein sequence databases could discover new biological mechanisms and targets in AML. However, proteogenomics workflows including bioinformatics guidelines can be challenging for the wide AML research community. It is expected that further automation and simplification of the bioinformatics procedures might attract AML investigators to adopt the proteogenomics strategy.

iProphet: Multi-level Integrative Analysis of Shotgun Proteomic Data Improves Peptide and Protein Identification Rates and Error Estimates*

PubMed Central

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

Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

PubMed

Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

2015-04-21

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

Improvement of Quantitative Measurements in Multiplex Proteomics Using High-Field Asymmetric Waveform Spectrometry.

PubMed

Pfammatter, Sibylle; Bonneil, Eric; Thibault, Pierre

2016-12-02

Quantitative proteomics using isobaric reagent tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provides a convenient approach to compare changes in protein abundance across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reductions in interfering ions, which increased the number of quantifiable peptides by up to 68% while significantly improving the accuracy of abundance measurements compared to that with conventional LC-MS/MS. The improvement in quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.

Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

PubMed Central

Schlautman, Joshua D; Rozek, Wojciech; Stetler, Robert; Mosley, R Lee; Gendelman, Howard E; Ciborowski, Pawel

2008-01-01

Background The ProteomeLab™ PF 2D platform is a relatively new approach to global protein profiling. Herein, it was used for investigation of plasma proteome changes in amyotrophic lateral sclerosis (ALS) patients before and during immunization with glatiramer acetate (GA) in a clinical trial. Results The experimental design included immunoaffinity depletion of 12 most abundant proteins from plasma samples with the ProteomeLab™ IgY-12 LC10 column kit as first dimension separation, also referred to as immuno-partitioning. Second and third dimension separations of the enriched proteome were performed on the PF 2D platform utilizing 2D isoelectric focusing and RP-HPLC with the resulting fractions collected for analysis. 1D gel electrophoresis was added as a fourth dimension when sufficient protein was available. Protein identification from collected fractions was performed using nano-LC-MS/MS approach. Analysis of differences in the resulting two-dimensional maps of fractions obtained from the PF 2D and the ability to identify proteins from these fractions allowed sensitivity threshold measurements. Masked proteins in the PF 2D fractions are discussed. Conclusion We offer some insight into the strengths and limitations of this emerging proteomic platform. PMID:18789151

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

PubMed

Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics.

PubMed

Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L

2010-07-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Development of an open source laboratory information management system for 2-D gel electrophoresis-based proteomics workflow

PubMed Central

Morisawa, Hiraku; Hirota, Mikako; Toda, Tosifusa

2006-01-01

Background In the post-genome era, most research scientists working in the field of proteomics are confronted with difficulties in management of large volumes of data, which they are required to keep in formats suitable for subsequent data mining. Therefore, a well-developed open source laboratory information management system (LIMS) should be available for their proteomics research studies. Results We developed an open source LIMS appropriately customized for 2-D gel electrophoresis-based proteomics workflow. The main features of its design are compactness, flexibility and connectivity to public databases. It supports the handling of data imported from mass spectrometry software and 2-D gel image analysis software. The LIMS is equipped with the same input interface for 2-D gel information as a clickable map on public 2DPAGE databases. The LIMS allows researchers to follow their own experimental procedures by reviewing the illustrations of 2-D gel maps and well layouts on the digestion plates and MS sample plates. Conclusion Our new open source LIMS is now available as a basic model for proteome informatics, and is accessible for further improvement. We hope that many research scientists working in the field of proteomics will evaluate our LIMS and suggest ways in which it can be improved. PMID:17018156

Systematic Errors in Peptide and Protein Identification and Quantification by Modified Peptides*

PubMed Central

Bogdanow, Boris; Zauber, Henrik; Selbach, Matthias

2016-01-01

The principle of shotgun proteomics is to use peptide mass spectra in order to identify corresponding sequences in a protein database. The quality of peptide and protein identification and quantification critically depends on the sensitivity and specificity of this assignment process. Many peptides in proteomic samples carry biochemical modifications, and a large fraction of unassigned spectra arise from modified peptides. Spectra derived from modified peptides can erroneously be assigned to wrong amino acid sequences. However, the impact of this problem on proteomic data has not yet been investigated systematically. Here we use combinations of different database searches to show that modified peptides can be responsible for 20–50% of false positive identifications in deep proteomic data sets. These false positive hits are particularly problematic as they have significantly higher scores and higher intensities than other false positive matches. Furthermore, these wrong peptide assignments lead to hundreds of false protein identifications and systematic biases in protein quantification. We devise a “cleaned search” strategy to address this problem and show that this considerably improves the sensitivity and specificity of proteomic data. In summary, we show that modified peptides cause systematic errors in peptide and protein identification and quantification and should therefore be considered to further improve the quality of proteomic data annotation. PMID:27215553

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

PubMed Central

Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

2010-01-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

Microbial Interactions in Plants: Perspectives and Applications of Proteomics.

PubMed

Imam, Jahangir; Shukla, Pratyoosh; Mandal, Nimai Prasad; Variar, Mukund

2017-01-01

The structure and function of proteins involved in plant-microbe interactions is investigated through large-scale proteomics technology in a complex biological sample. Since the whole genome sequences are now available for several plant species and microbes, proteomics study has become easier, accurate and huge amount of data can be generated and analyzed during plant-microbe interactions. Proteomics approaches are highly important and relevant in many studies and showed that only genomics approaches are not sufficient enough as much significant information are lost as the proteins and not the genes coding them are final product that is responsible for the observed phenotype. Novel approaches in proteomics are developing continuously enabling the study of the various aspects in arrangements and configuration of proteins and its functions. Its application is becoming more common and frequently used in plant-microbe interactions with the advancement in new technologies. They are more used for the portrayal of cell and extracellular destructiveness and pathogenicity variables delivered by pathogens. This distinguishes the protein level adjustments in host plants when infected with pathogens and advantageous partners. This review provides a brief overview of different proteomics technology which is currently available followed by their exploitation to study the plant-microbe interaction. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Combining Search Engines for Comparative Proteomics

PubMed Central

Tabb, David

2012-01-01

Many proteomics laboratories have found spectral counting to be an ideal way to recognize biomarkers that differentiate cohorts of samples. This approach assumes that proteins that differ in quantity between samples will generate different numbers of identifiable tandem mass spectra. Increasingly, researchers are employing multiple search engines to maximize the identifications generated from data collections. This talk evaluates four strategies to combine information from multiple search engines in comparative proteomics. The “Count Sum” model pools the spectra across search engines. The “Vote Counting” model combines the judgments from each search engine by protein. Two other models employ parametric and non-parametric analyses of protein-specific p-values from different search engines. We evaluated the four strategies in two different data sets. The ABRF iPRG 2009 study generated five LC-MS/MS analyses of “red” E. coli and five analyses of “yellow” E. coli. NCI CPTAC Study 6 generated five concentrations of Sigma UPS1 spiked into a yeast background. All data were identified with X!Tandem, Sequest, MyriMatch, and TagRecon. For both sample types, “Vote Counting” appeared to manage the diverse identification sets most effectively, yielding heightened discrimination as more search engines were added.

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

PubMed

Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective classification and detection of malignant, premalignant and healthy conditions. The method is extremely sensitive to detect proteins with limit of detection of the order of femto-moles. The HPLC-LIF combined with PCA as a potential proteomic method for the diagnosis of oral cancer and cervical cancer has been discussed in this paper. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography—Tandem Mass Spectrometry

PubMed Central

Tabb, David L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Variyath, Asokan Mulayath; Ham, Amy-Joan L.; Bunk, David M.; Kilpatrick, Lisa E.; Billheimer, Dean D.; Blackman, Ronald K.; Cardasis, Helene L.; Carr, Steven A.; Clauser, Karl R.; Jaffe, Jacob D.; Kowalski, Kevin A.; Neubert, Thomas A.; Regnier, Fred E.; Schilling, Birgit; Tegeler, Tony J.; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fisher, Susan J.; Gibson, Bradford W.; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Stein, Steven E.; Tempst, Paul; Paulovich, Amanda G.; Liebler, Daniel C.; Spiegelman, Cliff

2009-01-01

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35–60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies. PMID:19921851

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio)

PubMed Central

Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V.; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine. PMID:29145436

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio).

PubMed

Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Parsing and Quantification of Raw Orbitrap Mass Spectrometer Data Using RawQuant.

PubMed

Kovalchik, Kevin A; Moggridge, Sophie; Chen, David D Y; Morin, Gregg B; Hughes, Christopher S

2018-06-01

Effective analysis of protein samples by mass spectrometry (MS) requires careful selection and optimization of a range of experimental parameters. As the output from the primary detection device, the "raw" MS data file can be used to gauge the success of a given sample analysis. However, the closed-source nature of the standard raw MS file can complicate effective parsing of the data contained within. To ease and increase the range of analyses possible, the RawQuant tool was developed to enable parsing of raw MS files derived from Thermo Orbitrap instruments to yield meta and scan data in an openly readable text format. RawQuant can be commanded to export user-friendly files containing MS 1 , MS 2 , and MS 3 metadata as well as matrices of quantification values based on isobaric tagging approaches. In this study, the utility of RawQuant is demonstrated in several scenarios: (1) reanalysis of shotgun proteomics data for the identification of the human proteome, (2) reanalysis of experiments utilizing isobaric tagging for whole-proteome quantification, and (3) analysis of a novel bacterial proteome and synthetic peptide mixture for assessing quantification accuracy when using isobaric tags. Together, these analyses successfully demonstrate RawQuant for the efficient parsing and quantification of data from raw Thermo Orbitrap MS files acquired in a range of common proteomics experiments. In addition, the individual analyses using RawQuant highlights parametric considerations in the different experimental sets and suggests targetable areas to improve depth of coverage in identification-focused studies and quantification accuracy when using isobaric tags.

Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome*

PubMed Central

Tholen, Stefan; Biniossek, Martin L.; Gansz, Martina; Gomez-Auli, Alejandro; Bengsch, Fee; Noel, Agnes; Kizhakkedathu, Jayachandran N.; Boerries, Melanie; Busch, Hauke; Reinheckel, Thomas; Schilling, Oliver

2013-01-01

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb−/−, and Ctsl−/− mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl−/− skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl−/− skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl−/− or Ctsb−/− samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably. PMID:23233448

Proteomics in the Classroom: An Investigative Study of Proteins in Microorganisms

ERIC Educational Resources Information Center

Benskin, Jon; Chen, Sixue

2012-01-01

As advances in biotechnology and molecular biology rapidly expand in research settings, it is vital that we continue to prepare high school students to enter and thrive in those modern laboratories. This multistep, inquiry-based lab describes highly adaptable methods to teach students not only current molecular techniques and technologies, but…

A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data

PubMed Central

Chen, Yi-Hau

2017-01-01

Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https://github.com/roqe/T2GA. PMID:28622336

A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data.

PubMed

Lai, En-Yu; Chen, Yi-Hau; Wu, Kun-Pin

2017-06-01

Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https://github.com/roqe/T2GA.

Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections

PubMed Central

2014-01-01

The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways. PMID:25071420

Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

PubMed Central

Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; McClelland, Michael; Heffron, Fred; Smith, Richard D.

2009-01-01

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella. PMID:19277208

Effects of three commonly-used diuretics on the urinary proteome.

PubMed

Li, Xundou; Zhao, Mindi; Li, Menglin; Jia, Lulu; Gao, Youhe

2014-06-01

Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F; hydrochlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the criteria of P≤0.05, a fold change ≥2, a spectral count ≥5, and false positive rate (FDR) ≤1%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC-MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics. Copyright © 2014. Production and hosting by Elsevier Ltd.

Proteomic analysis of Medulloblastoma reveals functional biology with translational potential.

PubMed

Rivero-Hinojosa, Samuel; Lau, Ling San; Stampar, Mojca; Staal, Jerome; Zhang, Huizhen; Gordish-Dressman, Heather; Northcott, Paul A; Pfister, Stefan M; Taylor, Michael D; Brown, Kristy J; Rood, Brian R

2018-06-07

Genomic characterization has begun to redefine diagnostic classifications of cancers. However, it remains a challenge to infer disease phenotypes from genomic alterations alone. To help realize the promise of genomics, we have performed a quantitative proteomics investigation using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and 41 tissue samples spanning the 4 genomically based subgroups of medulloblastoma and control cerebellum. We have identified and quantitated thousands of proteins across these groups and find that we are able to recapitulate the genomic subgroups based upon subgroup restricted and differentially abundant proteins while also identifying subgroup specific protein isoforms. Integrating our proteomic measurements with genomic data, we calculate a poor correlation between mRNA and protein abundance. Using EPIC 850 k methylation array data on the same tissues, we also investigate the influence of copy number alterations and DNA methylation on the proteome in an attempt to characterize the impact of these genetic features on the proteome. Reciprocally, we are able to use the proteome to identify which genomic alterations result in altered protein abundance and thus are most likely to impact biology. Finally, we are able to assemble protein-based pathways yielding potential avenues for clinical intervention. From these, we validate the EIF4F cap-dependent translation pathway as a novel druggable pathway in medulloblastoma. Thus, quantitative proteomics complements genomic platforms to yield a more complete understanding of functional tumor biology and identify novel therapeutic targets for medulloblastoma.

Intrinsic circannual regulation of brown adipose tissue form and function in tune with hibernation

PubMed Central

Hindle, Allyson G.

2013-01-01

Winter hibernators repeatedly cycle between cold torpor and rewarming supported by nonshivering thermogenesis in brown adipose tissue (BAT). In contrast, summer animals are homeotherms, undergoing reproduction, growth, and fattening. This life history confers variability to BAT recruitment and activity. To address the components underlying prewinter enhancement and winter activation, we interrogated the BAT proteome in 13-lined ground squirrels among three summer and five winter states. We also examined mixed physiology in fall and spring individuals to test for ambient temperature and seasonal effects, as well as the timing of seasonal transitions. BAT form and function differ circannually in these animals, as evidenced by morphology and proteome dynamics. This intrinsic pattern distinguished homeothermic groups and early vs. late winter hibernators. Homeothermic variation derived from postemergence delay in growth and substrate biosynthesis. The heterothermic proteome varied less despite extreme winter physiological shifts and was optimized to exploit lipids by enhanced fatty acid binding, β-oxidation, and mitochondrial protein translocation. Surprisingly, ambient temperature did not affect the BAT proteome during transition seasons; rather, the pronounced summer-winter shift preceded environmental changes and phenotypic progression. During fall transition, differential regulation of two fatty acid binding proteins provides further evidence of recruitment and separates proteomic preparation from successful hibernation. Abundance of FABP4 correlates with torpor bout length throughout the year, clarifying its potential function in hibernation. Metabolically active BAT is a target for treating human obesity and metabolic disorders. Understanding the hibernator's extreme and seasonally distinct recruitment and activation control strategies offers untapped potential to identify novel, therapeutically relevant regulatory pathways. PMID:24326419

Assessing signal-to-noise in quantitative proteomics: multivariate statistical analysis in DIGE experiments.

PubMed

Friedman, David B

2012-01-01

All quantitative proteomics experiments measure variation between samples. When performing large-scale experiments that involve multiple conditions or treatments, the experimental design should include the appropriate number of individual biological replicates from each condition to enable the distinction between a relevant biological signal from technical noise. Multivariate statistical analyses, such as principal component analysis (PCA), provide a global perspective on experimental variation, thereby enabling the assessment of whether the variation describes the expected biological signal or the unanticipated technical/biological noise inherent in the system. Examples will be shown from high-resolution multivariable DIGE experiments where PCA was instrumental in demonstrating biologically significant variation as well as sample outliers, fouled samples, and overriding technical variation that would not be readily observed using standard univariate tests.

Free Flow Zonal Electrophoresis for Fractionation of Plant Membrane Compartments Prior to Proteomic Analysis.

PubMed

Barkla, Bronwyn J

2018-01-01

Free flow zonal electrophoresis (FFZE) is a versatile, reproducible, and potentially high-throughput technique for the separation of plant organelles and membranes by differences in membrane surface charge. It offers considerable benefits over traditional fractionation techniques, such as density gradient centrifugation and two-phase partitioning, as it is relatively fast, sample recovery is high, and the method provides unparalleled sample purity. It has been used to successfully purify chloroplasts and mitochondria from plants but also, to obtain highly pure fractions of plasma membrane, tonoplast, ER, Golgi, and thylakoid membranes. Application of the technique can significantly improve protein coverage in large-scale proteomics studies by decreasing sample complexity. Here, we describe the method for the fractionation of plant cellular membranes from leaves by FFZE.

Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety “Westin”

PubMed Central

Dória, Milena Santos; de Sousa, Aurizângela Oliveira; Barbosa, Cristiane de Jesus; Costa, Márcio Gilberto Cardoso; Gesteira, Abelmon da Silva; Souza, Regina Martins; Freitas, Ana Camila Oliveira; Pirovani, Carlos Priminho

2015-01-01

Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus. PMID:26207751

Citrus tristeza virus (CTV) Causing Proteomic and Enzymatic Changes in Sweet Orange Variety "Westin".

PubMed

Dória, Milena Santos; Sousa, Aurizângela Oliveira de; Barbosa, Cristiane de Jesus; Costa, Márcio Gilberto Cardoso; Gesteira, Abelmon da Silva; Souza, Regina Martins; Freitas, Ana Camila Oliveira; Pirovani, Carlos Priminho

2015-01-01

Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.

Proteomics analysis of BHK-21 cells infected with a fixed strain of rabies virus.

PubMed

Zandi, Fatemeh; Eslami, Naser; Soheili, Masoomeh; Fayaz, Ahmad; Gholami, Alireza; Vaziri, Behrouz

2009-05-01

Rabies is a neurotropic virus that causes a life threatening acute viral encephalitis. The complex relationship of rabies virus (RV) with the host leads to its replication and spreading toward the neural network, where viral pathogenic effects appeared as neuronal dysfunction. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with challenge virus standard strain of RV was performed. This cell line is an in vitro model for rabies infection and is commonly used for viral seed preparation. The direct effect of the virus on cellular protein machinery was investigated by 2-DE proteome mapping of infected versus control cells followed by LC-MS/MS identification. This analysis revealed significant changes in expression of 14 proteins, seven of these proteins were viral and the remaining were host proteins with different known functions: cytoskeletal (capping protein, vimentin), anti-oxidative stress (superoxide dismutase), regulatory (Stathmin), and protein synthesis (P0). Despite of limited changes appeared upon rabies infection, they present a set of interesting biochemical pathways for further investigation on viral-host interaction.

DMSO Assisted Electrospray Ionization for the Detection of Small Peptide Hormones in Urine by Dilute-and-Shoot-Liquid-Chromatography-High Resolution Mass Spectrometry

NASA Astrophysics Data System (ADS)

Judák, Péter; Grainger, Janelle; Goebel, Catrin; Van Eenoo, Peter; Deventer, Koen

2017-08-01

The mobile phase additive (DMSO) has been described as a useful tool to enhance electrospray ionization (ESI) of peptides and proteins. So far, this technique has mainly been used in proteomic/peptide research, and its applicability in a routine clinical laboratory setting (i.e., doping control analysis) has not been described yet. This work provides a simple, easy to implement screening method for the detection of doping relevant small peptides (GHRPs, GnRHs, GHS, and vasopressin-analogues) with molecular weight less than 2 kDa applying DMSO in the mobile phase. The gain in sensitivity was sufficient to inject the urine samples after a 2-fold dilution step omitting a time consuming sample preparation. The employed analytical procedure was validated for the qualitative determination of 36 compounds, including 13 metabolites. The detection limits (LODs) ranged between 50 and 1000 pg/mL and were compliant with the 2 ng/mL minimum detection level required by the World Anti-Doping Agency (WADA) for all the target peptides. To demonstrate the feasibility of the work, urine samples obtained from patients who have been treated with desmopressin or leuprolide and urine samples that have been declared as adverse analytical findings were analyzed.

Application of Large-Scale Aptamer-Based Proteomic Profiling to Planned Myocardial Infarctions.

PubMed

Jacob, Jaison; Ngo, Debby; Finkel, Nancy; Pitts, Rebecca; Gleim, Scott; Benson, Mark D; Keyes, Michelle J; Farrell, Laurie A; Morgan, Thomas; Jennings, Lori L; Gerszten, Robert E

2018-03-20

Emerging proteomic technologies using novel affinity-based reagents allow for efficient multiplexing with high-sample throughput. To identify early biomarkers of myocardial injury, we recently applied an aptamer-based proteomic profiling platform that measures 1129 proteins to samples from patients undergoing septal alcohol ablation for hypertrophic cardiomyopathy, a human model of planned myocardial injury. Here, we examined the scalability of this approach using a markedly expanded platform to study a far broader range of human proteins in the context of myocardial injury. We applied a highly multiplexed, expanded proteomic technique that uses single-stranded DNA aptamers to assay 4783 human proteins (4137 distinct human gene targets) to derivation and validation cohorts of planned myocardial injury, individuals with spontaneous myocardial infarction, and at-risk controls. We found 376 target proteins that significantly changed in the blood after planned myocardial injury in a derivation cohort (n=20; P <1.05E-05, 1-way repeated measures analysis of variance, Bonferroni threshold). Two hundred forty-seven of these proteins were validated in an independent planned myocardial injury cohort (n=15; P <1.33E-04, 1-way repeated measures analysis of variance); >90% were directionally consistent and reached nominal significance in the validation cohort. Among the validated proteins that were increased within 1 hour after planned myocardial injury, 29 were also elevated in patients with spontaneous myocardial infarction (n=63; P <6.17E-04). Many of the novel markers identified in our study are intracellular proteins not previously identified in the peripheral circulation or have functional roles relevant to myocardial injury. For example, the cardiac LIM protein, cysteine- and glycine-rich protein 3, is thought to mediate cardiac mechanotransduction and stress responses, whereas the mitochondrial ATP synthase F 0 subunit component is a vasoactive peptide on its release from cells. Last, we performed aptamer-affinity enrichment coupled with mass spectrometry to technically verify aptamer specificity for a subset of the new biomarkers. Our results demonstrate the feasibility of large-scale aptamer multiplexing at a level that has not previously been reported and with sample throughput that greatly exceeds other existing proteomic methods. The expanded aptamer-based proteomic platform provides a unique opportunity for biomarker and pathway discovery after myocardial injury. © 2017 American Heart Association, Inc.

Protein extraction method for the proteomic study of a Mexican traditional fermented starchy food.

PubMed

Cárdenas, C; Barkla, B J; Wacher, C; Delgado-Olivares, L; Rodríguez-Sanoja, R

2014-12-05

Pozol is a traditional fermented maize dough prepared in southeastern Mexico. Wide varieties of microorganisms have already been isolated from this spontaneously fermented product; and include fungi, yeasts, and lactic- and non-lactic acid bacteria. Pozol presents physicochemical features different from that of other food fermentation products, such as a high starch content, in addition to a low protein content. It is these qualities that make it intractable for protein recovery and characterization. The aim of this study was to develop a methodology to optimize the recovery of proteins from the pozol dough following fermentation, by reducing the complexity of the mixture prior to 2D-PAGE analysis and sequencing, to allow the characterization of the metaproteome of the dough. The proteome of 15day fermented maize dough was characterized; proteins were separated and analyzed by mass spectrometry (LC-MS/MS). Subsequent sequence homology database searching, identified numerous bacterial and fungi proteins; with a predominance of lactic acid bacterial proteins, mainly from the Lactobacillus genus. Fungi are mainly represented by Aspergillus. For dominant genera, the most prevalent proteins belong to carbohydrate metabolism and energy production, which suggest that at 15days of fermentation not only fungi but also bacteria are metabolically active. Several methodologies have been employed to study pozol, with a specific focus toward the identification of the microbiota of this fermented maize dough, using both traditional cultivation techniques and culture independent molecular techniques. However to date, the dynamics of this complex fermentation is not well understood. With the purpose to gain further insight into the nature of the fermentation, we used proteomic technologies to identify the origin of proteins and enzymes that facilitate substrate utilization and ultimately the development of the microbiota and fermentation. In this paper we overcome the first general challenge for such studies, obtaining a protein sample with adequate quality capable of representing the system. Copyright © 2014 Elsevier B.V. All rights reserved.

General method for rapid purification of native chromatin fragments.

PubMed

Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A; Denu, John M

2018-05-24

Biochemical, proteomic and epigenetic studies of chromatin rely on the efficient ability to isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state, and is applicable for both parallel multi-sample spin-column purification and large scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes, features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy.  The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies, and as a mild, acid- and detergent-free sample preparation method for mass-spectrometry analysis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

An Internal Standard for Assessing Phosphopeptide Recovery from Metal Ion/Oxide Enrichment Strategies

NASA Astrophysics Data System (ADS)

Paulo, Joao A.; Navarrete-Perea, Jose; Erickson, Alison R.; Knott, Jeffrey; Gygi, Steven P.

2018-04-01

Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. However, proteins with roles in these pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment, and subsequent recovery of phosphorylated peptides, is vital. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. We designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy. [Figure not available: see fulltext.

Extensive T-Cell Epitope Repertoire Sharing among Human Proteome, Gastrointestinal Microbiome, and Pathogenic Bacteria: Implications for the Definition of Self

PubMed Central

Bremel, Robert D.; Homan, E. Jane

2015-01-01

T-cell receptor binding to MHC-bound peptides plays a key role in discrimination between self and non-self. Only a subset, typically a pentamer, of amino acids in a MHC-bound peptide form the motif exposed to the T-cell receptor. We categorize and compare the T-cell exposed amino acid motif repertoire of the total proteomes of two groups of bacteria, comprising pathogens and gastrointestinal microbiome organisms, with the human proteome and immunoglobulins. Given the maximum 205, or 3.2 million of such motifs that bind T-cell receptors, there is considerable overlap in motif usage. We show that the human proteome, exclusive of immunoglobulins, only comprises three quarters of the possible motifs, of which 65.3% are also present in both composite bacterial proteomes. Very few motifs are unique to the human proteome. Immunoglobulin variable regions carry a broad diversity of T-cell exposed motifs (TCEMs) that provides a stratified random sample of the motifs found in pathogens, microbiome, and the human proteome. Individual bacterial genera and species vary in the content of immunoglobulin and human proteome matched motifs that they carry. Mycobacteria and Burkholderia spp carry a particularly high content of such matched motifs. Some bacteria retain a unique motif signature and motif sharing pattern with the human proteome. The implication is that distinguishing self from non-self does not depend on individual TCEMs, but on a complex and dynamic overlay of signals wherein the same TCEM may play different roles in different organisms, and the frequency with which a particular TCEM appears influences its effect. The patterns observed provide clues to bacterial immune evasion and to strategies for intervention, including vaccine design. The breadth and distinct frequency patterns of the immunoglobulin-derived peptides suggest a role of immunoglobulins in maintaining a broadly responsive T-cell repertoire. PMID:26557118

Phylogenetic Tracings of Proteome Size Support the Gradual Accretion of Protein Structural Domains and the Early Origin of Viruses from Primordial Cells

PubMed Central

Nasir, Arshan; Kim, Kyung Mo; Caetano-Anollés, Gustavo

2017-01-01

Untangling the origin and evolution of viruses remains a challenging proposition. We recently studied the global distribution of protein domain structures in thousands of completely sequenced viral and cellular proteomes with comparative genomics, phylogenomics, and multidimensional scaling methods. A tree of life describing the evolution of proteomes revealed viruses emerging from the base of the tree as a fourth supergroup of life. A tree of domains indicated an early origin of modern viral lineages from ancient cells that co-existed with the cellular ancestors. However, it was recently argued that the rooting of our trees and the basal placement of viruses was artifactually induced by small genome (proteome) size. Here we show that these claims arise from misunderstanding and misinterpretations of cladistic methodology. Trees are reconstructed unrooted, and thus, their topologies cannot be distorted a posteriori by the rooting methodology. Tracing proteome size in trees and multidimensional views of evolutionary relationships as well as tests of leaf stability and exclusion/inclusion of taxa demonstrated that the smallest proteomes were neither attracted toward the root nor caused any topological distortions of the trees. Simulations confirmed that taxa clustering patterns were independent of proteome size and were determined by the presence of known evolutionary relatives in data matrices, highlighting the need for broader taxon sampling in phylogeny reconstruction. Instead, phylogenetic tracings of proteome size revealed a slowdown in innovation of the structural domain vocabulary and four regimes of allometric scaling that reflected a Heaps law. These regimes explained increasing economies of scale in the evolutionary growth and accretion of kernel proteome repertoires of viruses and cellular organisms that resemble growth of human languages with limited vocabulary sizes. Results reconcile dynamic and static views of domain frequency distributions that are consistent with the axiom of spatiotemporal continuity that is tenet of evolutionary thinking. PMID:28690608

Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

PubMed

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.

Advances in Quantitative Proteomics of Microbes and Microbial Communities

NASA Astrophysics Data System (ADS)

Waldbauer, J.; Zhang, L.; Rizzo, A. I.

2015-12-01

Quantitative measurements of gene expression are key to developing a mechanistic, predictive understanding of how microbial metabolism drives many biogeochemical fluxes and responds to environmental change. High-throughput RNA-sequencing can afford a wealth of information about transcript-level expression patterns, but it is becoming clear that expression dynamics are often very different at the protein level where biochemistry actually occurs. These divergent dynamics between levels of biological organization necessitate quantitative proteomic measurements to address many biogeochemical questions. The protein-level expression changes that underlie shifts in the magnitude, or even the direction, of metabolic and biogeochemical fluxes can be quite subtle and test the limits of current quantitative proteomics techniques. Here we describe methodologies for high-precision, whole-proteome quantification that are applicable to both model organisms of biogeochemical interest that may not be genetically tractable, and to complex community samples from natural environments. Employing chemical derivatization of peptides with multiple isotopically-coded tags, this strategy is rapid and inexpensive, can be implemented on a wide range of mass spectrometric instrumentation, and is relatively insensitive to chromatographic variability. We demonstrate the utility of this quantitative proteomics approach in application to both isolates and natural communities of sulfur-metabolizing and photosynthetic microbes.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

PubMed

Wada, Yoshinao; Dell, Anne; Haslam, Stuart M; Tissot, Bérangère; Canis, Kévin; Azadi, Parastoo; Bäckström, Malin; Costello, Catherine E; Hansson, Gunnar C; Hiki, Yoshiyuki; Ishihara, Mayumi; Ito, Hiromi; Kakehi, Kazuaki; Karlsson, Niclas; Hayes, Catherine E; Kato, Koichi; Kawasaki, Nana; Khoo, Kay-Hooi; Kobayashi, Kunihiko; Kolarich, Daniel; Kondo, Akihiro; Lebrilla, Carlito; Nakano, Miyako; Narimatsu, Hisashi; Novak, Jan; Novotny, Milos V; Ohno, Erina; Packer, Nicolle H; Palaima, Elizabeth; Renfrow, Matthew B; Tajiri, Michiko; Thomsson, Kristina A; Yagi, Hirokazu; Yu, Shin-Yi; Taniguchi, Naoyuki

2010-04-01

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Biomarkers of Aspergillus spores

NASA Astrophysics Data System (ADS)

Sulc, Miroslav; Peslova, Katerina; Zabka, Martin; Hajduch, Marian; Havlicek, Vladimir

2009-02-01

We applied both matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric and 1D sodium dodecylsulfate polyacrylamide gel electrophoretic (1D-PAGE) approaches for direct analysis of intact fungal spores of twenty four Aspergillus species. In parallel, we optimized various protocols for protein extraction from Aspergillus spores using acidic conditions, step organic gradient and variable sonication treatment. The MALDI-TOF mass spectra obtained from optimally prepared samples provided a reproducible fingerprint demonstrating the capability of the MALDI-TOF approach to type and characterize different fungal strains within the Aspergillus genus. Mass spectra of intact fungal spores provided signals mostly below 20 kDa. The minimum material amount represented 0.3 [mu]g (10,000 spores). Proteins with higher molecular weight were detected by 1D-PAGEE Eleven proteins were identified from three selected strains in the range 5-25 kDa by the proteomic approach. Hemolysin and hydrophobin have the highest relevance in host-pathogen interactions.

Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

PubMed

Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

2013-04-05

To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

Recent advances and opportunities in proteomic analyses of tumour heterogeneity.

PubMed

Bateman, Nicholas W; Conrads, Thomas P

2018-04-01

Solid tumour malignancies comprise a highly variable admixture of tumour and non-tumour cellular populations, forming a complex cellular ecosystem and tumour microenvironment. This tumour heterogeneity is not incidental, and is known to correlate with poor patient prognosis for many cancer types. Indeed, non-malignant cell populations, such as vascular endothelial and immune cells, are known to play key roles supporting and, in some cases, driving aggressive tumour biology, and represent targets of emerging therapeutics, such as antiangiogenesis and immune checkpoint inhibitors. The biochemical interplay between these cellular populations and how they contribute to molecular tumour heterogeneity remains enigmatic, particularly from the perspective of the tumour proteome. This review focuses on recent advances in proteomic methods, namely imaging mass spectrometry, single-cell proteomic techniques, and preanalytical sample processing, that are uniquely positioned to enable detailed analysis of discrete cellular populations within tumours to improve our understanding of tumour proteomic heterogeneity. This review further emphasizes the opportunity afforded by the application of these techniques to the analysis of tumour heterogeneity in formalin-fixed paraffin-embedded archival tumour tissues, as these represent an invaluable resource for retrospective analyses that is now routinely accessible, owing to recent technological and methodological advances in tumour tissue proteomics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of the rat cerebrospinal fluid proteome following acute cerebral ischemia using an aptamer-based proteomic technology.

PubMed

Simats, Alba; García-Berrocoso, Teresa; Ramiro, Laura; Giralt, Dolors; Gill, Natalia; Penalba, Anna; Bustamante, Alejandro; Rosell, Anna; Montaner, Joan

2018-05-21

The limited accessibility to the brain has turned the cerebrospinal fluid (CSF) into a valuable source that may contribute to the complete understanding of the stroke pathophysiology. Here we have described the CSF proteome in the hyper-acute phase of cerebral ischemia by performing an aptamer-based proteomic assay (SOMAscan) in CSF samples collected before and 30 min after male Wistar rats had undergone a 90 min Middle Cerebral Artery Occlusion (MCAO) or sham-surgery. Proteomic results indicated that cerebral ischemia acutely increased the CSF levels of 716 proteins, mostly overrepresented in leukocyte chemotaxis and neuronal death processes. Seven promising candidates were further evaluated in rat plasma and brain (CKB, CaMK2A, CaMK2B, CaMK2D, PDXP, AREG, CMPK). The 3 CaMK2 family-members and CMPK early decreased in the infarcted brain area and, together with AREG, co-localized with neurons. Conversely, CKB levels remained consistent after the insult and specifically matched with astrocytes. Further exploration of these candidates in human plasma revealed the potential of CKB and CMPK to diagnose stroke, while CaMK2B and CMPK resulted feasible biomarkers of functional stroke outcome. Our findings provided insights into the CSF proteome following cerebral ischemia and identified new outstanding proteins that might be further considered as potential biomarkers of stroke.

Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation

PubMed Central

Zhang, Qiang; Cundiff, Judy K.; Maria, Sarah D.; McMahon, Robert J.; Woo, Jessica G.; Davidson, Barbara S.; Morrow, Ardythe L.

2013-01-01

In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study. PMID:28250401

Advances in targeted proteomics and applications to biomedical research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

PubMed Central

Clement, Cristina C.; Aphkhazava, David; Nieves, Edward; Callaway, Myrasol; Olszewski, Waldemar; Rotzschke, Olaf; Santambrogio, Laura

2013-01-01

In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC–MS–MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell–cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids. PMID:23202415

MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles.

PubMed

Lauterbach, Alexander; Usbeck, Julia C; Behr, Jürgen; Vogel, Rudi F

2017-01-01

Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own "in-house strains". During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable "brewing yeast" spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking.

MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles

PubMed Central

Lauterbach, Alexander; Usbeck, Julia C.; Behr, Jürgen

2017-01-01

Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own “in-house strains”. During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization–Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable “brewing yeast” spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking. PMID:28792944

Proteomic analysis of urine in patients with intestinal segments transposed into the urinary tract.

PubMed

Nabi, Ghulam; N'Dow, James; Hasan, Tahseen S; Booth, Ian R; Cash, Phil

2005-04-01

Intestinal segments are used to replace or reconstruct the urinary bladder when it has become dysfunctional or develops life-threatening disease such as cancer. The quality of life in patients with intestinal segments used to either enlarge or completely replace the native bladder is adversely affected by recurrent urinary tract infections, excessive mucus production and the occasional development of malignancy. At present, there is no reliable method of predicting or noninvasively monitoring these patients for the development of these complications. The characterisation of proteins secreted into urine from the transposed intestinal segments could serve as important indicators of these clinical complications. Urine is an ideal source of material in which to search for biomarkers, since it bathes the affected tissues and can be obtained relatively easily by noninvasive methods. The urinary proteome of patients with intestinal segments transposed into the urinary tract is unknown and we present the first global description of the urinary protein profile in these patients. Sample preparation is a critical step in achieving accurate and reliable data. We describe a method to prepare urinary proteins that was compatible with their subsequent analysis using two-dimensional polyacrylamide gel electrophoresis. This method helped to overcome some of the technical problems encountered in analysing urine from this patient cohort. The method was used to analyse urinary proteins recovered from five healthy controls and ten patients with intestinal segments transposed into the urinary tract. Four low molecular weight proteins were found to be present in nine out of ten for the patient group but for none of the healthy controls. The four proteins were identified as lithostathine-1 alpha precursor, pancreatitis associated protein-1 precursor, liver fatty acid binding protein and testis expressed protein-12. The role of these proteins as potential biomarkers of intestinal cell activity within the reconstructed bladder is discussed.

A liquid chromatography-tandem mass spectrometry-based targeted proteomics assay for monitoring P-glycoprotein levels in human breast tissue.

PubMed

Yang, Ting; Chen, Fei; Xu, Feifei; Wang, Fengliang; Xu, Qingqing; Chen, Yun

2014-09-25

P-glycoprotein (P-gp) can efflux drugs from cancer cells, and its overexpression is commonly associated with multi-drug resistance (MDR). Thus, the accurate quantification of P-gp would help predict the response to chemotherapy and for prognosis of breast cancer patients. An advanced liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics assay was developed and validated for monitoring P-gp levels in breast tissue. Tryptic peptide 368IIDNKPSIDSYSK380 was selected as a surrogate analyte for quantification, and immuno-depleted tissue extract was used as a surrogate matrix. Matched pairs of breast tissue samples from 60 patients who were suspected to have drug resistance were subject to analysis. The levels of P-gp were quantified. Using data from normal tissue, we suggested a P-gp reference interval. The experimental values of tumor tissue samples were compared with those obtained from Western blotting and immunohistochemistry (IHC). The result indicated that the targeted proteomics approach was comparable to IHC but provided a lower limit of quantification (LOQ) and could afford more reliable results at low concentrations than the other two methods. LC/MS/MS-based targeted proteomics may allow the quantification of P-gp in breast tissue in a more accurate manner. Copyright © 2014 Elsevier B.V. All rights reserved.