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Sample records for accurate protein quantification

  1. The SILAC Fly Allows for Accurate Protein Quantification in Vivo*

    PubMed Central

    Sury, Matthias D.; Chen, Jia-Xuan; Selbach, Matthias

    2010-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these “SILAC flies” to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sex-specific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics. PMID:20525996

  2. Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (ZIA).

    PubMed

    Vesterberg, O

    1994-05-01

    For zone immunoelectrophoresis assay (ZIA) glass tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35-150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol-binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.

  3. A novel automated image analysis method for accurate adipocyte quantification

    PubMed Central

    Osman, Osman S; Selway, Joanne L; Kępczyńska, Małgorzata A; Stocker, Claire J; O’Dowd, Jacqueline F; Cawthorne, Michael A; Arch, Jonathan RS; Jassim, Sabah; Langlands, Kenneth

    2013-01-01

    Increased adipocyte size and number are associated with many of the adverse effects observed in metabolic disease states. While methods to quantify such changes in the adipocyte are of scientific and clinical interest, manual methods to determine adipocyte size are both laborious and intractable to large scale investigations. Moreover, existing computational methods are not fully automated. We, therefore, developed a novel automatic method to provide accurate measurements of the cross-sectional area of adipocytes in histological sections, allowing rapid high-throughput quantification of fat cell size and number. Photomicrographs of H&E-stained paraffin sections of murine gonadal adipose were transformed using standard image processing/analysis algorithms to reduce background and enhance edge-detection. This allowed the isolation of individual adipocytes from which their area could be calculated. Performance was compared with manual measurements made from the same images, in which adipocyte area was calculated from estimates of the major and minor axes of individual adipocytes. Both methods identified an increase in mean adipocyte size in a murine model of obesity, with good concordance, although the calculation used to identify cell area from manual measurements was found to consistently over-estimate cell size. Here we report an accurate method to determine adipocyte area in histological sections that provides a considerable time saving over manual methods. PMID:23991362

  4. Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification

    PubMed Central

    Weisbrod, Chad R.; Eng, Jimmy K.; Hoopmann, Michael R.; Baker, Tahmina; Bruce, James E.

    2012-01-01

    FT All Reaction Monitoring (FT-ARM) is a novel approach for the identification and quantification of peptides that relies upon the selectivity of high mass accuracy data and the specificity of peptide fragmentation patterns. An FT-ARM experiment involves continuous, data-independent, high mass accuracy MS/MS acquisition spanning a defined m/z range. Custom software was developed to search peptides against the multiplexed fragmentation spectra by comparing theoretical or empirical fragment ions against every fragmentation spectrum across the entire acquisition. A dot product score is calculated against each spectrum in order to generate a score chromatogram used for both identification and quantification. Chromatographic elution profile characteristics are not used to cluster precursor peptide signals to their respective fragment ions. FT-ARM identifications are demonstrated to be complementary to conventional data-dependent shotgun analysis, especially in cases where the data-dependent method fails due to fragmenting multiple overlapping precursors. The sensitivity, robustness and specificity of FT-ARM quantification are shown to be analogous to selected reaction monitoring-based peptide quantification with the added benefit of minimal assay development. Thus, FT-ARM is demonstrated to be a novel and complementary data acquisition, identification, and quantification method for the large scale analysis of peptides. PMID:22288382

  5. Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ *

    PubMed Central

    Cox, Jürgen; Hein, Marco Y.; Luber, Christian A.; Paron, Igor; Nagaraj, Nagarjuna; Mann, Matthias

    2014-01-01

    Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity determination and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein separation prior to LC-MS analysis. Protein abundance profiles are assembled using the maximum possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technology that is readily applicable to many biological questions; it is compatible with standard statistical analysis workflows, and it has been validated in many and diverse biological projects. Our algorithms can handle very large experiments of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button. PMID:24942700

  6. Neutron-Encoded Protein Quantification by Peptide Carbamylation

    NASA Astrophysics Data System (ADS)

    Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

    2014-01-01

    We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

  7. Accurate Quantification of Disease Markers in Human Serum Using Iron Oxide Nanoparticle-linked Immunosorbent Assay

    PubMed Central

    Zhang, Linlin; Tong, Sheng; Zhou, Jun; Bao, Gang

    2016-01-01

    Accurate and reliable quantification of biomarkers in the blood is essential in disease screening and diagnosis. Here we describe an iron oxide nanoparticle (IONP)-linked immunosorbent assay (ILISA) for detecting biomolecules in human serum. Sandwich ILISA was optimized for the detection of four important serological markers, IgA, IgG, IgM, and C-reactive protein (CRP), and assessed with normal sera, simulated disease-state sera and the serum samples from patients infected with West Nile virus (WNV) or human herpes virus (HHV). Our study shows that using the detection assay formulated with 18.8 nm wüstite nanocrystals, ILISA can achieve sub-picomolar detection sensitivity, and all four markers can be accurately quantified over a large dynamic range. In addition, ILISA is not susceptible to variations in operating procedures and shows better linearity and higher stability compared with ELISA, which facilitates its integration into detection methods suitable for point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications. PMID:27375784

  8. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  9. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-01

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described. PMID:25411902

  10. Proximity assays for sensitive quantification of proteins.

    PubMed

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S; Bustin, Stephen A

    2015-06-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein-protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  11. Proximity assays for sensitive quantification of proteins

    PubMed Central

    Greenwood, Christina; Ruff, David; Kirvell, Sara; Johnson, Gemma; Dhillon, Harvinder S.; Bustin, Stephen A.

    2015-01-01

    Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression. PMID:27077033

  12. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    Introduction In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). Conclusions 2-LTR circles

  13. Simple and accurate quantification of quantum dots via single-particle counting.

    PubMed

    Zhang, Chun-yang; Johnson, Lawrence W

    2008-03-26

    Quantification of quantum dots (QDs) is essential to the quality control of QD synthesis, development of QD-based LEDs and lasers, functionalizing of QDs with biomolecules, and engineering of QDs for biological applications. However, simple and accurate quantification of QD concentration in a variety of buffer solutions and in complex mixtures still remains a critical technological challenge. Here, we introduce a new methodology for quantification of QDs via single-particle counting, which is conceptually different from established UV-vis absorption and fluorescence spectrum techniques where large amounts of purified QDs are needed and specific absorption coefficient or quantum yield values are necessary for measurements. We demonstrate that single-particle counting allows us to nondiscriminately quantify different kinds of QDs by their distinct fluorescence burst counts in a variety of buffer solutions regardless of their composition, structure, and surface modifications, and without the necessity of absorption coefficient and quantum yield values. This single-particle counting can also unambiguously quantify individual QDs in a complex mixture, which is practically impossible for both UV-vis absorption and fluorescence spectrum measurements. Importantly, the application of this single-particle counting is not just limited to QDs but also can be extended to fluorescent microspheres, quantum dot-based microbeads, and fluorescent nano rods, some of which currently lack efficient quantification methods.

  14. Simple and accurate quantification of BTEX in ambient air by SPME and GC-MS.

    PubMed

    Baimatova, Nassiba; Kenessov, Bulat; Koziel, Jacek A; Carlsen, Lars; Bektassov, Marat; Demyanenko, Olga P

    2016-07-01

    Benzene, toluene, ethylbenzene and xylenes (BTEX) comprise one of the most ubiquitous and hazardous groups of ambient air pollutants of concern. Application of standard analytical methods for quantification of BTEX is limited by the complexity of sampling and sample preparation equipment, and budget requirements. Methods based on SPME represent simpler alternative, but still require complex calibration procedures. The objective of this research was to develop a simpler, low-budget, and accurate method for quantification of BTEX in ambient air based on SPME and GC-MS. Standard 20-mL headspace vials were used for field air sampling and calibration. To avoid challenges with obtaining and working with 'zero' air, slope factors of external standard calibration were determined using standard addition and inherently polluted lab air. For polydimethylsiloxane (PDMS) fiber, differences between the slope factors of calibration plots obtained using lab and outdoor air were below 14%. PDMS fiber provided higher precision during calibration while the use of Carboxen/PDMS fiber resulted in lower detection limits for benzene and toluene. To provide sufficient accuracy, the use of 20mL vials requires triplicate sampling and analysis. The method was successfully applied for analysis of 108 ambient air samples from Almaty, Kazakhstan. Average concentrations of benzene, toluene, ethylbenzene and o-xylene were 53, 57, 11 and 14µgm(-3), respectively. The developed method can be modified for further quantification of a wider range of volatile organic compounds in air. In addition, the new method is amenable to automation. PMID:27154647

  15. Fast MS/MS acquisition without dynamic exclusion enables precise and accurate quantification of proteome by MS/MS fragment intensity

    PubMed Central

    Zhang, Shen; Wu, Qi; Shan, Yichu; Zhao, Qun; Zhao, Baofeng; Weng, Yejing; Sui, Zhigang; Zhang, Lihua; Zhang, Yukui

    2016-01-01

    Most currently proteomic studies use data-dependent acquisition with dynamic exclusion to identify and quantify the peptides generated by the digestion of biological sample. Although dynamic exclusion permits more identifications and higher possibility to find low abundant proteins, stochastic and irreproducible precursor ion selection caused by dynamic exclusion limit the quantification capabilities, especially for MS/MS based quantification. This is because a peptide is usually triggered for fragmentation only once due to dynamic exclusion. Therefore the fragment ions used for quantification only reflect the peptide abundances at that given time point. Here, we propose a strategy of fast MS/MS acquisition without dynamic exclusion to enable precise and accurate quantification of proteome by MS/MS fragment intensity. The results showed comparable proteome identification efficiency compared to the traditional data-dependent acquisition with dynamic exclusion, better quantitative accuracy and reproducibility regardless of label-free based quantification or isobaric labeling based quantification. It provides us with new insights to fully explore the potential of modern mass spectrometers. This strategy was applied to the relative quantification of two human disease cell lines, showing great promises for quantitative proteomic applications. PMID:27198003

  16. Accurate joint space quantification in knee osteoarthritis: a digital x-ray tomosynthesis phantom study

    NASA Astrophysics Data System (ADS)

    Sewell, Tanzania S.; Piacsek, Kelly L.; Heckel, Beth A.; Sabol, John M.

    2011-03-01

    The current imaging standard for diagnosis and monitoring of knee osteoarthritis (OA) is projection radiography. However radiographs may be insensitive to markers of early disease such as osteophytes and joint space narrowing (JSN). Relative to standard radiography, digital X-ray tomosynthesis (DTS) may provide improved visualization of the markers of knee OA without the interference of superimposed anatomy. DTS utilizes a series of low-dose projection images over an arc of +/-20 degrees to reconstruct tomographic images parallel to the detector. We propose that DTS can increase accuracy and precision in JSN quantification. The geometric accuracy of DTS was characterized by quantifying joint space width (JSW) as a function of knee flexion and position using physical and anthropomorphic phantoms. Using a commercially available digital X-ray system, projection and DTS images were acquired for a Lucite rod phantom with known gaps at various source-object-distances, and angles of flexion. Gap width, representative of JSW, was measured using a validated algorithm. Over an object-to-detector-distance range of 5-21cm, a 3.0mm gap width was reproducibly measured in the DTS images, independent of magnification. A simulated 0.50mm (+/-0.13) JSN was quantified accurately (95% CI 0.44-0.56mm) in the DTS images. Angling the rods to represent knee flexion, the minimum gap could be precisely determined from the DTS images and was independent of flexion angle. JSN quantification using DTS was insensitive to distance from patient barrier and flexion angle. Potential exists for the optimization of DTS for accurate radiographic quantification of knee OA independent of patient positioning.

  17. Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2014-12-17

    Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate.

  18. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  19. Biomarker Quantification in Unprocessed Human Sera by Using A New Nanomagnetic Protein Assay

    NASA Astrophysics Data System (ADS)

    Li, Yuanpeng; Jing, Ying; Srinivasan, Balasubramanian; Yao, Xiaofeng; Hugger, Marie A.; Xing, Chengguo; Wang, Jian-Ping

    2010-12-01

    Early recognition and prevention of chronic disease, such as lung cancer, require a fast, accurate detection and longitudinal monitoring on potential biomarkers, which could identify the molecule change in the initial stage of the chronic disease. Here we report the realization of specific and accurate quantification of a low-abundance serum protein in unprocessed human sera, using our novel giant magnetoresistive (GMR) biosensing system with uniform high-magnetic-moment nanoparticles and a competition based detection scheme. Only one antibody is needed for such detection scheme. The quantification of interleukin-6 (IL-6, a low-abundance protein and a potential cancer biomarker), as low as 125 fM IL-6 proteins, directly in 4 μL of unprocessed human sera was demonstrated within 5 minutes by such system. The results nicely differentiate normal individuals and lung cancer patients. This platform has great potential to facilitate the identification and validation of disease biomarkers.

  20. Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins.

    PubMed

    She, Yi-Min; Rosu-Myles, Michael; Walrond, Lisa; Cyr, Terry D

    2012-02-01

    Mass spectrometry (MS)-based quantification of highly homologous proteins in complex samples has proven difficult due to subtle sequence variations and the wide dynamic range of protein isoforms present. Herein, we report the use of reductive dimethylation on intact proteins to quantitatively compare protein isoform expression in the nucleus and cytoplasm of mesenchymal stem cells (MSC) and normal stroma. By coupling fixed-charge MS/MS scanning, high-resolution UPLC FT-MS data-dependent acquisition and MASCOT-based data mining, hydrogen/deuterium-labeled dimethyl-lysine peptides were simultaneously captured allowing the accurate comparison of 123 protein isoforms in parallel LC MS/MS runs. Thirty-four isoforms were identified that had expression levels specific to MSC. Where possible, proteomic analyses were verified by Western blotting and were demonstrated to be divergent from the level of gene transcription detected for certain proteins. Our analysis provides a protein isoform signature specific to MSC and demonstrates the suitability of dimethyl-lysine labeling on intact proteins for quantifying highly homologous proteins on a proteome-wide scale.

  1. Nanomagnetic competition assay for low-abundance protein biomarker quantification in unprocessed human sera.

    PubMed

    Li, Yuanpeng; Srinivasan, Balasubramanian; Jing, Ying; Yao, Xiaofeng; Hugger, Marie A; Wang, Jian-Ping; Xing, Chengguo

    2010-03-31

    A novel giant magnetoresistive sensor and uniform high-magnetic-moment FeCo nanoparticles (12.8 nm)-based detecting platform with minimized detecting distance was developed for rapid biomolecule quantification from body fluids. Such a system demonstrates specific, accurate, and quick detection and quantification of interleukin-6, a low-abundance protein and a potential cancer biomarker, directly in 4 muL of unprocessed human sera. This platform is expected to facilitate the identification and validation of disease biomarkers. It may eventually lead to a low-cost personal medical device for chronic disease early detection, diagnosis, and prognosis.

  2. ACCURATE QUANTIFICATION OF DRIED RESIDUE THIN FILMS USING X-RAY FLUORESCENCE

    SciTech Connect

    C. WORLEY; G. HAVRILLA

    2000-09-01

    An XRF specimen preparation method was developed to quantify the concentration of gallium in plutonium metal while minimizing the risk of contaminating the instrument with radioactive material. To ensure that homogeneous specimens are examined, plutonium is dissolved in dilute HCl and HNO{sub 3} prior to analysis. In the preliminary work here, non-radioactive aqueous gallium standards were prepared, and zinc was added as an internal standard to improve the accuracy and precision. Aliquots from these solutions were cast on Mylar XRF films and air dried prior to analysis. Two methods of casting the solutions were evaluated: (1) casting as a thin layer using a surfactant to wet the support film and (2) casting multiple small spots on the support film. Aqueous gallium standards were prepared and cast as dried residue specimens using each method. These specimens were then analyzed, and calibration curves were prepared. Highly linear calibrations were obtained for each preparation method when zinc was used as the internal standard (RMS values {le}1% of the standards concentration range in both cases). Based on this preliminary work, this dried residue process appears very promising for the accurate quantification of gallium in plutonium.

  3. Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins

    PubMed Central

    Sanz-Hernández, Máximo; Vostrikov, Vitaly V.; Veglia, Gianluigi; De Simone, Alfonso

    2016-01-01

    The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that are often responsible for the biological function of oligomeric membrane protein states. PMID:26975211

  4. Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication.

    PubMed

    Valekova, Ivona; Skalnikova, Helena Kupcova; Jarkovska, Karla; Motlik, Jan; Kovarova, Hana

    2015-01-01

    Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners). PMID:25063502

  5. Quantification of protein concentration using UV absorbance and Coomassie dyes.

    PubMed

    Noble, James E

    2014-01-01

    The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used. PMID:24423263

  6. Label-Free Identification and Quantification of SUMO Target Proteins.

    PubMed

    Hendriks, Ivo A; Vertegaal, Alfred C O

    2016-01-01

    Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days. PMID:27631806

  7. New approach for the quantification of processed animal proteins in feed using light microscopy.

    PubMed

    Veys, P; Baeten, V

    2010-07-01

    A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed. PMID:20432096

  8. Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method.

    PubMed

    Lee, Naery; Shin, SukJin; Chung, Hye Joo; Kim, Do Keun; Lim, Jong-Mi; Park, Hyunsung; Oh, Ho Jung

    2015-09-22

    Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents. We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories. PMID:26275477

  9. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  10. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  11. Dielectrophoretic immobilization of proteins: Quantification by atomic force microscopy.

    PubMed

    Laux, Eva-Maria; Knigge, Xenia; Bier, Frank F; Wenger, Christian; Hölzel, Ralph

    2015-09-01

    The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, for example the applied voltage and duration of field application, are varied systematically, and the influence on the amount of immobilized proteins is investigated. A linear correlation to the duration of field application was found by atomic force microscopy, and both microscopical methods yield a square dependence of the amount of immobilized proteins on the applied voltage. While fluorescence microscopy allows real-time imaging, atomic force microscopy reveals immobilized proteins obscured in fluorescence images due to low S/N. Furthermore, the higher spatial resolution of the atomic force microscope enables the visualization of the protein distribution on single nanoelectrodes. The electric field distribution is calculated and compared to experimental results with very good agreement to atomic force microscopy measurements.

  12. Gas-phase purification enables accurate, large-scale, multiplexed proteome quantification with isobaric tagging

    PubMed Central

    Wenger, Craig D; Lee, M Violet; Hebert, Alexander S; McAlister, Graeme C; Phanstiel, Douglas H; Westphall, Michael S; Coon, Joshua J

    2011-01-01

    We describe a mass spectrometry method, QuantMode, which improves the accuracy of isobaric tag–based quantification by alleviating the pervasive problem of precursor interference—co-isolation of impurities—through gas-phase purification. QuantMode analysis of a yeast sample ‘contaminated’ with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan, with a small loss of spectral identifications. This technique will allow large-scale, multiplexed quantitative proteomics analyses using isobaric tagging. PMID:21963608

  13. An on-bacterium flow cytometric immunoassay for protein quantification.

    PubMed

    Lan, Wen-Jun; Lan, Wei; Wang, Hai-Yan; Yan, Lei; Wang, Zhe-Li

    2013-09-01

    The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein. PMID:23739299

  14. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  15. Relative Quantification of Several Plasma Proteins during Liver Transplantation Surgery

    PubMed Central

    Parviainen, Ville; Joenväärä, Sakari; Tukiainen, Eija; Ilmakunnas, Minna; Isoniemi, Helena; Renkonen, Risto

    2011-01-01

    Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery. PMID:22187521

  16. Relative quantification of several plasma proteins during liver transplantation surgery.

    PubMed

    Parviainen, Ville; Joenväärä, Sakari; Tukiainen, Eija; Ilmakunnas, Minna; Isoniemi, Helena; Renkonen, Risto

    2011-01-01

    Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50-2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.

  17. Three-dimensional dynamic contrast-enhanced MRI for the accurate, extensive quantification of microvascular permeability in atherosclerotic plaques.

    PubMed

    Calcagno, Claudia; Lobatto, Mark E; Dyvorne, Hadrien; Robson, Philip M; Millon, Antoine; Senders, Max L; Lairez, Olivier; Ramachandran, Sarayu; Coolen, Bram F; Black, Alexandra; Mulder, Willem J M; Fayad, Zahi A

    2015-10-01

    Atherosclerotic plaques that cause stroke and myocardial infarction are characterized by increased microvascular permeability and inflammation. Dynamic contrast-enhanced MRI (DCE-MRI) has been proposed as a method to quantify vessel wall microvascular permeability in vivo. Until now, most DCE-MRI studies of atherosclerosis have been limited to two-dimensional (2D) multi-slice imaging. Although providing the high spatial resolution required to image the arterial vessel wall, these approaches do not allow the quantification of plaque permeability with extensive anatomical coverage, an essential feature when imaging heterogeneous diseases, such as atherosclerosis. To our knowledge, we present the first systematic evaluation of three-dimensional (3D), high-resolution, DCE-MRI for the extensive quantification of plaque permeability along an entire vascular bed, with validation in atherosclerotic rabbits. We compare two acquisitions: 3D turbo field echo (TFE) with motion-sensitized-driven equilibrium (MSDE) preparation and 3D turbo spin echo (TSE). We find 3D TFE DCE-MRI to be superior to 3D TSE DCE-MRI in terms of temporal stability metrics. Both sequences show good intra- and inter-observer reliability, and significant correlation with ex vivo permeability measurements by Evans Blue near-infrared fluorescence (NIRF). In addition, we explore the feasibility of using compressed sensing to accelerate 3D DCE-MRI of atherosclerosis, to improve its temporal resolution and therefore the accuracy of permeability quantification. Using retrospective under-sampling and reconstructions, we show that compressed sensing alone may allow the acceleration of 3D DCE-MRI by up to four-fold. We anticipate that the development of high-spatial-resolution 3D DCE-MRI with prospective compressed sensing acceleration may allow for the more accurate and extensive quantification of atherosclerotic plaque permeability along an entire vascular bed. We foresee that this approach may allow for

  18. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR.

    PubMed

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2015-02-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation.

  19. Accurate and Reliable Quantification of Total Microalgal Fuel Potential as Fatty Acid Methyl Esters by in situ Transesterfication

    SciTech Connect

    Laurens, L. M. L.; Quinn, M.; Van Wychen, S.; Templeton, D. W.; Wolfrum, E. J.

    2012-04-01

    In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4-7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.

  20. Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification.

    PubMed

    Laurens, Lieve M L; Quinn, Matthew; Van Wychen, Stefanie; Templeton, David W; Wolfrum, Edward J

    2012-04-01

    In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4-7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process. PMID:22349344

  1. Statistically accurate low-order models for uncertainty quantification in turbulent dynamical systems

    PubMed Central

    Sapsis, Themistoklis P.; Majda, Andrew J.

    2013-01-01

    A framework for low-order predictive statistical modeling and uncertainty quantification in turbulent dynamical systems is developed here. These reduced-order, modified quasilinear Gaussian (ROMQG) algorithms apply to turbulent dynamical systems in which there is significant linear instability or linear nonnormal dynamics in the unperturbed system and energy-conserving nonlinear interactions that transfer energy from the unstable modes to the stable modes where dissipation occurs, resulting in a statistical steady state; such turbulent dynamical systems are ubiquitous in geophysical and engineering turbulence. The ROMQG method involves constructing a low-order, nonlinear, dynamical system for the mean and covariance statistics in the reduced subspace that has the unperturbed statistics as a stable fixed point and optimally incorporates the indirect effect of non-Gaussian third-order statistics for the unperturbed system in a systematic calibration stage. This calibration procedure is achieved through information involving only the mean and covariance statistics for the unperturbed equilibrium. The performance of the ROMQG algorithm is assessed on two stringent test cases: the 40-mode Lorenz 96 model mimicking midlatitude atmospheric turbulence and two-layer baroclinic models for high-latitude ocean turbulence with over 125,000 degrees of freedom. In the Lorenz 96 model, the ROMQG algorithm with just a single mode captures the transient response to random or deterministic forcing. For the baroclinic ocean turbulence models, the inexpensive ROMQG algorithm with 252 modes, less than 0.2% of the total, captures the nonlinear response of the energy, the heat flux, and even the one-dimensional energy and heat flux spectra. PMID:23918398

  2. Detection and quantification of protein-protein interactions by far-western blotting.

    PubMed

    Jadwin, Joshua A; Mayer, Bruce J; Machida, Kazuya

    2015-01-01

    Far-western blotting is a convenient method to characterize protein-protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. When specific modular protein binding domains are used as probes, this approach allows characterization of protein-protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

  3. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  4. Conditional mutual inclusive information enables accurate quantification of associations in gene regulatory networks.

    PubMed

    Zhang, Xiujun; Zhao, Juan; Hao, Jin-Kao; Zhao, Xing-Ming; Chen, Luonan

    2015-03-11

    Mutual information (MI), a quantity describing the nonlinear dependence between two random variables, has been widely used to construct gene regulatory networks (GRNs). Despite its good performance, MI cannot separate the direct regulations from indirect ones among genes. Although the conditional mutual information (CMI) is able to identify the direct regulations, it generally underestimates the regulation strength, i.e. it may result in false negatives when inferring gene regulations. In this work, to overcome the problems, we propose a novel concept, namely conditional mutual inclusive information (CMI2), to describe the regulations between genes. Furthermore, with CMI2, we develop a new approach, namely CMI2NI (CMI2-based network inference), for reverse-engineering GRNs. In CMI2NI, CMI2 is used to quantify the mutual information between two genes given a third one through calculating the Kullback-Leibler divergence between the postulated distributions of including and excluding the edge between the two genes. The benchmark results on the GRNs from DREAM challenge as well as the SOS DNA repair network in Escherichia coli demonstrate the superior performance of CMI2NI. Specifically, even for gene expression data with small sample size, CMI2NI can not only infer the correct topology of the regulation networks but also accurately quantify the regulation strength between genes. As a case study, CMI2NI was also used to reconstruct cancer-specific GRNs using gene expression data from The Cancer Genome Atlas (TCGA). CMI2NI is freely accessible at http://www.comp-sysbio.org/cmi2ni.

  5. Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

    PubMed

    Randrianjatovo, I; Girbal-Neuhauser, E; Marcato-Romain, C-E

    2015-06-01

    Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml(-1)) showed little or no sugar interference up to 2000 μg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

  6. Cytofluorometric Quantification of Cell Death Elicited by NLR Proteins.

    PubMed

    Sica, Valentina; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2016-01-01

    Nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins, also known as NOD-like receptors, are critical components of the molecular machinery that senses intracellular danger signals to initiate an innate immune response against invading pathogens or endogenous sources of hazard. The best characterized effect of NLR signaling is the secretion of various cytokines with immunostimulatory effects, including interleukin (IL)-1β and IL-18. Moreover, at least under specific circumstances, NLRs can promote regulated variants of cell death. Here, we detail two protocols for the cytofluorometric quantification of cell death-associated parameters that can be conveniently employed to assess the lethal activity of specific NLRs or their ligands.

  7. Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; De Spiegelaere, Ward

    2014-01-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  8. Accurate quantification of tio2 nanoparticles collected on air filters using a microwave-assisted acid digestion method.

    PubMed

    Mudunkotuwa, Imali A; Anthony, T Renée; Grassian, Vicki H; Peters, Thomas M

    2016-01-01

    Titanium dioxide (TiO(2)) particles, including nanoparticles with diameters smaller than 100 nm, are used extensively in consumer products. In a 2011 current intelligence bulletin, the National Institute of Occupational Safety and Health (NIOSH) recommended methods to assess worker exposures to fine and ultrafine TiO(2) particles and associated occupational exposure limits for these particles. However, there are several challenges and problems encountered with these recommended exposure assessment methods involving the accurate quantitation of titanium dioxide collected on air filters using acid digestion followed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Specifically, recommended digestion methods include the use of chemicals, such as perchloric acid, which are typically unavailable in most accredited industrial hygiene laboratories due to highly corrosive and oxidizing properties. Other alternative methods that are used typically involve the use of nitric acid or combination of nitric acid and sulfuric acid, which yield very poor recoveries for titanium dioxide. Therefore, given the current state of the science, it is clear that a new method is needed for exposure assessment. In this current study, a microwave-assisted acid digestion method has been specifically designed to improve the recovery of titanium in TiO(2) nanoparticles for quantitative analysis using ICP-OES. The optimum digestion conditions were determined by changing several variables including the acids used, digestion time, and temperature. Consequently, the optimized digestion temperature of 210°C with concentrated sulfuric and nitric acid (2:1 v/v) resulted in a recovery of >90% for TiO(2). The method is expected to provide for a more accurate quantification of airborne TiO(2) particles in the workplace environment.

  9. Accurate quantification of tio2 nanoparticles collected on air filters using a microwave-assisted acid digestion method.

    PubMed

    Mudunkotuwa, Imali A; Anthony, T Renée; Grassian, Vicki H; Peters, Thomas M

    2016-01-01

    Titanium dioxide (TiO(2)) particles, including nanoparticles with diameters smaller than 100 nm, are used extensively in consumer products. In a 2011 current intelligence bulletin, the National Institute of Occupational Safety and Health (NIOSH) recommended methods to assess worker exposures to fine and ultrafine TiO(2) particles and associated occupational exposure limits for these particles. However, there are several challenges and problems encountered with these recommended exposure assessment methods involving the accurate quantitation of titanium dioxide collected on air filters using acid digestion followed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Specifically, recommended digestion methods include the use of chemicals, such as perchloric acid, which are typically unavailable in most accredited industrial hygiene laboratories due to highly corrosive and oxidizing properties. Other alternative methods that are used typically involve the use of nitric acid or combination of nitric acid and sulfuric acid, which yield very poor recoveries for titanium dioxide. Therefore, given the current state of the science, it is clear that a new method is needed for exposure assessment. In this current study, a microwave-assisted acid digestion method has been specifically designed to improve the recovery of titanium in TiO(2) nanoparticles for quantitative analysis using ICP-OES. The optimum digestion conditions were determined by changing several variables including the acids used, digestion time, and temperature. Consequently, the optimized digestion temperature of 210°C with concentrated sulfuric and nitric acid (2:1 v/v) resulted in a recovery of >90% for TiO(2). The method is expected to provide for a more accurate quantification of airborne TiO(2) particles in the workplace environment. PMID:26181824

  10. Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms.

    PubMed

    Wang, Xiaoli; Suo, Yongshan; Yin, Ruichuan; Shen, Heqing; Wang, Hailin

    2011-06-01

    Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50-5.70%. By using 2-deoxyguanosine as an internal standard, UPLC-MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC-MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.

  11. Quantification of protein interaction kinetics in a micro droplet

    SciTech Connect

    Yin, L. L.; Wang, S. P. E-mail: njtao@asu.edu; Shan, X. N.; Tao, N. J. E-mail: njtao@asu.edu; Zhang, S. T.

    2015-11-15

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  12. Quantification of protein interaction kinetics in a micro droplet

    NASA Astrophysics Data System (ADS)

    Yin, L. L.; Wang, S. P.; Shan, X. N.; Zhang, S. T.; Tao, N. J.

    2015-11-01

    Characterization of protein interactions is essential to the discovery of disease biomarkers, the development of diagnostic assays, and the screening for therapeutic drugs. Conventional flow-through kinetic measurements need relative large amount of sample that is not feasible for precious protein samples. We report a novel method to measure protein interaction kinetics in a single droplet with sub microliter or less volume. A droplet in a humidity-controlled environmental chamber is replacing the microfluidic channels as the reactor for the protein interaction. The binding process is monitored by a surface plasmon resonance imaging (SPRi) system. Association curves are obtained from the average SPR image intensity in the center area of the droplet. The washing step required by conventional flow-through SPR method is eliminated in the droplet method. The association and dissociation rate constants and binding affinity of an antigen-antibody interaction are obtained by global fitting of association curves at different concentrations. The result obtained by this method is accurate as validated by conventional flow-through SPR system. This droplet-based method not only allows kinetic studies for proteins with limited supply but also opens the door for high-throughput protein interaction study in a droplet-based microarray format that enables measurement of many to many interactions on a single chip.

  13. PROMALS web server for accurate multiple protein sequence alignments.

    PubMed

    Pei, Jimin; Kim, Bong-Hyun; Tang, Ming; Grishin, Nick V

    2007-07-01

    Multiple sequence alignments are essential in homology inference, structure modeling, functional prediction and phylogenetic analysis. We developed a web server that constructs multiple protein sequence alignments using PROMALS, a progressive method that improves alignment quality by using additional homologs from PSI-BLAST searches and secondary structure predictions from PSIPRED. PROMALS shows higher alignment accuracy than other advanced methods, such as MUMMALS, ProbCons, MAFFT and SPEM. The PROMALS web server takes FASTA format protein sequences as input. The output includes a colored alignment augmented with information about sequence grouping, predicted secondary structures and positional conservation. The PROMALS web server is available at: http://prodata.swmed.edu/promals/ PMID:17452345

  14. Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2015-08-01

    Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work has shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol ( m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo-1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications.

  15. Absolute protein quantification of the yeast chaperome under conditions of heat shock

    PubMed Central

    Mackenzie, Rebecca J.; Lawless, Craig; Holman, Stephen W.; Lanthaler, Karin; Beynon, Robert J.; Grant, Chris M.; Hubbard, Simon J.

    2016-01-01

    Chaperones are fundamental to regulating the heat shock response, mediating protein recovery from thermal‐induced misfolding and aggregation. Using the QconCAT strategy and selected reaction monitoring (SRM) for absolute protein quantification, we have determined copy per cell values for 49 key chaperones in Saccharomyces cerevisiae under conditions of normal growth and heat shock. This work extends a previous chemostat quantification study by including up to five Q‐peptides per protein to improve confidence in protein quantification. In contrast to the global proteome profile of S. cerevisiae in response to heat shock, which remains largely unchanged as determined by label‐free quantification, many of the chaperones are upregulated with an average two‐fold increase in protein abundance. Interestingly, eight of the significantly upregulated chaperones are direct gene targets of heat shock transcription factor‐1. By performing absolute quantification of chaperones under heat stress for the first time, we were able to evaluate the individual protein‐level response. Furthermore, this SRM data was used to calibrate label‐free quantification values for the proteome in absolute terms, thus improving relative quantification between the two conditions. This study significantly enhances the largely transcriptomic data available in the field and illustrates a more nuanced response at the protein level. PMID:27252046

  16. Reservoir evaluation of thin-bedded turbidites and hydrocarbon pore thickness estimation for an accurate quantification of resource

    NASA Astrophysics Data System (ADS)

    Omoniyi, Bayonle; Stow, Dorrik

    2016-04-01

    One of the major challenges in the assessment of and production from turbidite reservoirs is to take full account of thin and medium-bedded turbidites (<10cm and <30cm respectively). Although such thinner, low-pay sands may comprise a significant proportion of the reservoir succession, they can go unnoticed by conventional analysis and so negatively impact on reserve estimation, particularly in fields producing from prolific thick-bedded turbidite reservoirs. Field development plans often take little note of such thin beds, which are therefore bypassed by mainstream production. In fact, the trapped and bypassed fluids can be vital where maximising field value and optimising production are key business drivers. We have studied in detail, a succession of thin-bedded turbidites associated with thicker-bedded reservoir facies in the North Brae Field, UKCS, using a combination of conventional logs and cores to assess the significance of thin-bedded turbidites in computing hydrocarbon pore thickness (HPT). This quantity, being an indirect measure of thickness, is critical for an accurate estimation of original-oil-in-place (OOIP). By using a combination of conventional and unconventional logging analysis techniques, we obtain three different results for the reservoir intervals studied. These results include estimated net sand thickness, average sand thickness, and their distribution trend within a 3D structural grid. The net sand thickness varies from 205 to 380 ft, and HPT ranges from 21.53 to 39.90 ft. We observe that an integrated approach (neutron-density cross plots conditioned to cores) to HPT quantification reduces the associated uncertainties significantly, resulting in estimation of 96% of actual HPT. Further work will focus on assessing the 3D dynamic connectivity of the low-pay sands with the surrounding thick-bedded turbidite facies.

  17. A Strategy for Accurate Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine at CpG Sites Within Gene Promoter.

    PubMed

    Qui, Yiping; Yang, Qi; Sui, Fang; Lu, Rong; Dang, Siwen; Ji, Meiju; He, Nongyue; Shi, Bingyin; Hou, Peng

    2015-06-01

    5-Methylcytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. Traditional bisulfite-based DNA methylation analysis techniques have been widely used in the detection of 5mC. However, they can not discriminate 5hmC from 5mC, leading to overestimate 5mC levels. We here introduce a strategy, combination of selective oxidation and bisulfite pyrosequencing (BS-Pyroseq), for quantification of both 5mC and 5hmC at CpG sites within the promoters of CDH1, DAPK, RARβ and RUNX3 genes in a panel of cell lines and clinical samples. As expected, oxidative bisulfite pyrosequencing (oxBS-Pyroseq) assay decreased overall or site-specific methylation levels of three of these genes in most cell lines as compared with BS-Pyroseq assay. Similarly, decreased overall or site-specific methylation levels of DAPK, RARβ and RUNX3 genes in laryngeal, gastric and thyroid cancer and their matched normal tissues, respectively, were also found by a comparison between these two techniques, particularly in cancerous tissues. In addition, by using this combined strategy and hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) assay, we demonstrated that TET1 up-regulated DAPK expression through promoter demethylation. Collectively, this strategy is easy to establish and accurately discriminates and quantifies 5mC and 5hmC at CpG sites within selected gene promoters. PMID:26353591

  18. Automated gas-phase purification for accurate, multiplexed quantification on a stand-alone ion trap mass spectrometer

    PubMed Central

    Vincent, Catherine E.; Rensvold, Jarred W.; Westphall, Michael S.; Pagliarini, David J.; Coon, Joshua J.

    2012-01-01

    Isobaric tagging enables the acquisition of highly-multiplexed proteome quantification but is hindered by the pervasive problem of precursor interference. The elimination of co-isolated contaminants prior to reporter tag generation can be achieved through the use of gas-phase purification via proton transfer ion/ion reactions (QuantMode); however, the original QuantMode technique was implemented on the high resolution linear ion trap-Orbitrap hybrid mass spectrometer enabled with electron transfer dissociation (ETD). Here we extend this technology to stand-alone linear ion trap systems (trapQuantMode). Facilitated by the use of inlet beam-type activation (i.e., trapHCD) for production and observation of the low mass-to-charge reporter region, this scan sequence comprises three separate events to maximize peptide identifications, minimize duty cycle requirements, and increase quantitative accuracy, precision, and dynamic range. Significant improvements in quantitative accuracy were attained over standard methods when using trapQuantMode (trapQM) to analyze an interference model system comprising tryptic peptides of yeast that we contaminated with human peptides. Finally, we demonstrate practical benefits of this method by analysis of the proteomic changes that occur during mouse skeletal muscle myoblast differentiation. While trapQM’s reduced duty cycle led to the identification of fewer proteins than conventional operation (4,050 vs. 2,964), trapQM identified more significant differences (>1.5 fold, 1,362 vs 1,132, respectively; P<0.05) between the proteomes of undifferentiated myoblasts and differentiated myotubes and nearly ten-fold more differences with changes greater than 5-fold (96 vs. 12). We further show that our trapQM dataset is superior for identifying changes in protein abundance that are consistent with the metabolic and structural changes known to accompany myotube formation. PMID:23046161

  19. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering.

    PubMed

    Hill, Ryan C; Calle, Elizabeth A; Dzieciatkowska, Monika; Niklason, Laura E; Hansen, Kirk C

    2015-04-01

    The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.

  20. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering*

    PubMed Central

    Hill, Ryan C.; Calle, Elizabeth A.; Dzieciatkowska, Monika; Niklason, Laura E.; Hansen, Kirk C.

    2015-01-01

    The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. PMID:25660013

  1. PSEA-Quant: a protein set enrichment analysis on label-free and label-based protein quantification data.

    PubMed

    Lavallée-Adam, Mathieu; Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

    2014-12-01

    The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766

  2. Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2013-11-01

    Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

  3. MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

    PubMed

    Klett, Javier; Núñez-Salgado, Alfonso; Dos Santos, Helena G; Cortés-Cabrera, Álvaro; Perona, Almudena; Gil-Redondo, Rubén; Abia, David; Gago, Federico; Morreale, Antonio

    2012-09-11

    An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

  4. PET optimization for improved assessment and accurate quantification of {sup 90}Y-microsphere biodistribution after radioembolization

    SciTech Connect

    Martí-Climent, Josep M. Prieto, Elena; Elosúa, César; Rodríguez-Fraile, Macarena; Domínguez-Prado, Inés; Vigil, Carmen; García-Velloso, María J.; Arbizu, Javier; Peñuelas, Iván; Richter, José A.

    2014-09-15

    Purpose: {sup 90}Y-microspheres are widely used for the radioembolization of metastatic liver cancer or hepatocellular carcinoma and there is a growing interest for imaging {sup 90}Y-microspheres with PET. The aim of this study is to evaluate the performance of a current generation PET/CT scanner for {sup 90}Y imaging and to optimize the PET protocol to improve the assessment and the quantification of {sup 90}Y-microsphere biodistribution after radioembolization. Methods: Data were acquired on a Biograph mCT-TrueV scanner with time of flight (TOF) and point spread function (PSF) modeling. Spatial resolution was measured with a{sup 90}Y point source. Sensitivity was evaluated using the NEMA 70 cm line source filled with {sup 90}Y. To evaluate the count rate performance, {sup 90}Y vials with activity ranging from 3.64 to 0.035 GBq were measured in the center of the field of view (CFOV). The energy spectrum was evaluated. Image quality with different reconstructions was studied using the Jaszczak phantom containing six hollow spheres (diameters: 31.3, 28.1, 21.8, 16.1, 13.3, and 10.5 mm), filled with a 207 kBq/ml {sup 90}Y concentration and a 5:1 sphere-to-background ratio. Acquisition time was adjusted to simulate the quality of a realistic clinical PET acquisition of a patient treated with SIR-Spheres{sup ®}. The developed methodology was applied to ten patients after SIR-Spheres{sup ®} treatment acquiring a 10 min per bed PET. Results: The energy spectrum showed the{sup 90}Y bremsstrahlung radiation. The {sup 90}Y transverse resolution, with filtered backprojection reconstruction, was 4.5 mm in the CFOV and degraded to 5.0 mm at 10 cm off-axis. {sup 90}Y absolute sensitivity was 0.40 kcps/MBq in the center of the field of view. Tendency of true and random rates as a function of the {sup 90}Y activity could be accurately described using linear and quadratic models, respectively. Phantom studies demonstrated that, due to low count statistics in {sup 90}Y PET

  5. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  6. Quantification of protein posttranslational modifications using stable isotope and mass spectrometry I: principles and applications.

    PubMed

    Jiang, Xinzhao Grace; Apostol, Izydor; Luo, Quanzhou; Lewis, Jeffrey; Keener, Ronald; Luo, Shun; Jerums, Matthew; Zhang, Xin; Wypych, Jette; Huang, Gang

    2012-02-15

    With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where ¹⁵N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of ¹⁵N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm.

  7. Profiling and accurate quantification of Rubus ellagitannins and ellagic acid conjugates using direct UPLC-Q-TOF HDMS and HPLC-DAD analysis.

    PubMed

    Gasperotti, Mattia; Masuero, Domenico; Vrhovsek, Urska; Guella, Graziano; Mattivi, Fulvio

    2010-04-28

    Accurate quantification and structural characterization of ellagitannins and ellagic acid conjugates in food, beverages, and food supplements are essential starting points for studying their effect on human health. However, accuracy is hindered both by the lack of pure standard compounds and by methods that maintain the compounds in their native form, avoiding any chemical modification of the structure. The objective of this work was to develop a new method for the purification, chromatographic separation, and accurate quantification of ellagitannins and ellagic acid conjugates to provide thorough characterization of the diversity in composition of 11 Rubus cultivars grown in Trentino, Italy. As such, two major steps were required: (i) the isolation and purification (with associated detailed structural characterization and determination of their molar extinction coefficients) of sanguiin H-6 and lambertianin C, providing essential data for their use, together with ellagic acid, as external standards, and (ii) the determination of the chemical structure of 20 novel minor ellagitannins and 4 ellagic acid conjugates on the basis of their Q-TOF-HDMS and DAD spectra. This survey of ellagitannins and ellagic acid conjugates provides evidence for the existence of significant differences in the pattern between and within blackberry and raspberry cultivars. To our knowledge, this is the first paper that has combined detailed metabolite profiling with accurate quantification of the main ellagitannins in Rubus using their respective standards.

  8. Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Hyun-Seok; Heun Kim, Sook; Jeong, Ji-Seon; Lee, Yong-Moon; Yim, Yong-Hyeon

    2015-10-01

    An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

  9. Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB.

    PubMed

    Sugiman-Marangos, Seiji N; Weiss, Yoni M; Junop, Murray S

    2016-04-19

    Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

  10. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics

    PubMed Central

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research. PMID:27571061

  11. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics.

    PubMed

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research. PMID:27571061

  12. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics.

    PubMed

    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research.

  13. Sequential extractions: A new way for protein quantification-data from peanut allergens.

    PubMed

    Zhou, Ningling; Li, Wenying; Wu, Zhihua; Li, Xin; Yang, Anshu; Tong, Ping; Chen, Hongbing

    2015-09-01

    Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by the Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of them in solution. Extracting all of the contents is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein contents and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If all proteins can be extracted with sufficient extraction times, the protein contents in the peanut matrix can be calculated using a mathematical summation formula. This sum should be all proteins in the matrix. The five-step sequential extraction method can provide a means to quantify certain proteins in the matrix.

  14. Sequential extractions: A new way for protein quantification-data from peanut allergens.

    PubMed

    Zhou, Ningling; Li, Wenying; Wu, Zhihua; Li, Xin; Yang, Anshu; Tong, Ping; Chen, Hongbing

    2015-09-01

    Quantification of certain protein contents in the matrix is essential in protein analyses. The amount of total protein in the matrix can be determined by the Kjeldahl method. However, few methods can quantify certain protein contents in the matrix without extracting all of them in solution. Extracting all of the contents is difficult for proteins, especially relatively insoluble ones. A five-step sequential extraction method was developed for the quantification of certain proteins in defatted peanut flour based on the relationship between the extracted protein contents and the extraction times. The extracted proteins (i.e., total protein, Ara h 1, and Ara h 2) were quantitatively analyzed in each extraction of the same condition. An exponential equation was obtained between the extraction times and the respective amount of extracted protein as well as both the total protein and a particular protein. In particular, the amount of protein extracted each time can be a geometric sequence. If all proteins can be extracted with sufficient extraction times, the protein contents in the peanut matrix can be calculated using a mathematical summation formula. This sum should be all proteins in the matrix. The five-step sequential extraction method can provide a means to quantify certain proteins in the matrix. PMID:26026388

  15. Mass spectrometry-based absolute quantification reveals rhythmic variation of mouse circadian clock proteins.

    PubMed

    Narumi, Ryohei; Shimizu, Yoshihiro; Ukai-Tadenuma, Maki; Ode, Koji L; Kanda, Genki N; Shinohara, Yuta; Sato, Aya; Matsumoto, Katsuhiko; Ueda, Hiroki R

    2016-06-14

    Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation. PMID:27247408

  16. Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.

    PubMed

    Sanmartí-Espinal, Marta; Galve, Roger; Iavicoli, Patrizia; Persuy, Marie-Annick; Pajot-Augy, Edith; Marco, M-Pilar; Samitier, Josep

    2016-03-01

    Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

  17. Quantification of proteins on gold nanoparticles by combining MALDI-TOF MS and proteolysis

    NASA Astrophysics Data System (ADS)

    Ju, Soomi; Yeo, Woon-Seok

    2012-04-01

    Protein-coated nanoparticles have been used in many studies, including those related to drug delivery, disease diagnosis, therapeutics, and bioassays. The number and density of proteins on the particles’ surface are important parameters that need to be calculable in most applications. While quantification methods for two-dimensional surface-bound proteins are commonly found, only a few methods for the quantification of proteins on three-dimensional surfaces such as nanoparticles have been reported. In this paper, we report on a new method of quantifying proteins on nanoparticles using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). In this method, the nanoparticle-bound proteins are digested by trypsin and the resulting peptide fragments are analyzed by MALDI-TOF MS after the addition of an isotope-labeled internal standard (IS) which has the same sequence as a reference peptide of the surface-bound protein. Comparing the mass intensities between the reference peptide and the IS allows the absolute quantification of proteins on nanoparticles, because they have the same molecular milieu. As a model system, gold nanoparticles were examined using bovine serum albumin (BSA) as a coating protein. We believe that our strategy will be a useful tool that can provide researchers with quantitative information about the proteins on surfaces of three-dimensional materials.

  18. Quantification of proteins on gold nanoparticles by combining MALDI-TOF MS and proteolysis.

    PubMed

    Ju, Soomi; Yeo, Woon-Seok

    2012-04-01

    Protein-coated nanoparticles have been used in many studies, including those related to drug delivery, disease diagnosis, therapeutics, and bioassays. The number and density of proteins on the particles' surface are important parameters that need to be calculable in most applications. While quantification methods for two-dimensional surface-bound proteins are commonly found, only a few methods for the quantification of proteins on three-dimensional surfaces such as nanoparticles have been reported. In this paper, we report on a new method of quantifying proteins on nanoparticles using matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). In this method, the nanoparticle-bound proteins are digested by trypsin and the resulting peptide fragments are analyzed by MALDI-TOF MS after the addition of an isotope-labeled internal standard (IS) which has the same sequence as a reference peptide of the surface-bound protein. Comparing the mass intensities between the reference peptide and the IS allows the absolute quantification of proteins on nanoparticles, because they have the same molecular milieu. As a model system, gold nanoparticles were examined using bovine serum albumin (BSA) as a coating protein. We believe that our strategy will be a useful tool that can provide researchers with quantitative information about the proteins on surfaces of three-dimensional materials. PMID:22417878

  19. Protein binding of antimicrobials: methods for quantification and for investigation of its impact on bacterial killing.

    PubMed

    Beer, Jürgen; Wagner, Claudia Christina; Zeitlinger, Markus

    2009-03-01

    Plasma protein binding of antimicrobial agents is considered to be a key characteristic of antibiotics as it affects both their pharmacokinetics and pharmacodynamics. However, up to the present, no standard methods for measuring protein binding or for quantification of the influence of protein binding on antimicrobial activity exist. This short-coming has previously led to conflicting results on antibacterial activity of highly protein-bound antibiotics. The present review, therefore, set out to summarize (1) methods for quantification of protein binding, (2) microbiological growth media used for determination of the impact of protein binding on antimicrobial activity of antibiotics, and (3) different pharmacodynamic in vitro studies that are used in this context. The advantages and disadvantages of a wide range of different approaches are discussed and compared. The urgent call for international standardization by microbiological societies and laboratories may be considered as a logical consequence of the presented data. PMID:19117135

  20. Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification.

    PubMed

    Qin, Weijie; Song, Zifeng; Fan, Chao; Zhang, Wanjun; Cai, Yun; Zhang, Yangjun; Qian, Xiaohong

    2012-04-01

    In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin

  1. Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors.

    PubMed

    Vermeire, Jolien; Naessens, Evelien; Vanderstraeten, Hanne; Landi, Alessia; Iannucci, Veronica; Van Nuffel, Anouk; Taghon, Tom; Pizzato, Massimo; Verhasselt, Bruno

    2012-01-01

    Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.

  2. Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

    PubMed Central

    Vermeire, Jolien; Naessens, Evelien; Vanderstraeten, Hanne; Landi, Alessia; Iannucci, Veronica; Van Nuffel, Anouk; Taghon, Tom; Pizzato, Massimo; Verhasselt, Bruno

    2012-01-01

    Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories. PMID:23227216

  3. Quantification of detergent using colorimetric methods in membrane protein crystallography.

    PubMed

    Prince, Chelsy; Jia, Zongchao

    2015-01-01

    Membrane protein crystallography has the potential to greatly aid our understanding of membrane protein biology. Yet, membrane protein crystals remain challenging to produce. Although robust methods for the expression and purification of membrane proteins continue to be developed, the detergent component of membrane protein samples is equally important to crystallization efforts. This chapter describes the development of three colorimetric assays for the quantitation of detergent in membrane protein samples and provides detailed protocols. All of these techniques use small sample volumes and have potential applications in crystallography. The application of these techniques in crystallization prescreening, detergent concentration modification, and detergent exchange experiments is demonstrated. It has been observed that the concentration of detergent in a membrane protein sample can be just as important as the protein concentration when attempting to reproduce crystallization lead conditions.

  4. A rapid and accurate quantification method for real-time dynamic analysis of cellular lipids during microalgal fermentation processes in Chlorella protothecoides with low field nuclear magnetic resonance.

    PubMed

    Wang, Tao; Liu, Tingting; Wang, Zejian; Tian, Xiwei; Yang, Yi; Guo, Meijin; Chu, Ju; Zhuang, Yingping

    2016-05-01

    The rapid and real-time lipid determination can provide valuable information on process regulation and optimization in the algal lipid mass production. In this study, a rapid, accurate and precise quantification method of in vivo cellular lipids of Chlorella protothecoides using low field nuclear magnetic resonance (LF-NMR) was newly developed. LF-NMR was extremely sensitive to the algal lipids with the limits of the detection (LOD) of 0.0026g and 0.32g/L in dry lipid samples and algal broth, respectively, as well as limits of quantification (LOQ) of 0.0093g and 1.18g/L. Moreover, the LF-NMR signal was specifically proportional to the cellular lipids of C. protothecoides, thus the superior regression curves existing in a wide detection range from 0.02 to 0.42g for dry lipids and from 1.12 to 8.97gL(-1) of lipid concentration for in vivo lipid quantification were obtained with all R(2) higher than 0.99, irrespective of the lipid content and fatty acids profile variations. The accuracy of this novel method was further verified to be reliable by comparing lipid quantification results to those obtained by GC-MS. And the relative standard deviation (RSD) of LF-NMR results were smaller than 2%, suggesting the precision of this method. Finally, this method was successfully used in the on-line lipid monitoring during the algal lipid fermentation processes, making it possible for better understanding of the lipid accumulation mechanism and dynamic bioprocess control. PMID:26948045

  5. Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes

    PubMed Central

    2012-01-01

    Background Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. Results We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. Conclusions The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-β signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications. PMID:22248082

  6. Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies

    PubMed Central

    2013-01-01

    The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay (‘QFI-PCR’) that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing. PMID:24001039

  7. Protein Quantification by Elemental Mass Spectrometry: An Experiment for Graduate Students

    ERIC Educational Resources Information Center

    Schwarz, Gunnar; Ickert, Stefanie; Wegner, Nina; Nehring, Andreas; Beck, Sebastian; Tiemann, Ruediger; Linscheid, Michael W.

    2014-01-01

    A multiday laboratory experiment was designed to integrate inductively coupled plasma-mass spectrometry (ICP-MS) in the context of protein quantification into an advanced practical course in analytical and environmental chemistry. Graduate students were familiar with the analytical methods employed, whereas the combination of bioanalytical assays…

  8. Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance1[W

    PubMed Central

    Nikolovski, Nino; Shliaha, Pavel V.; Gatto, Laurent; Dupree, Paul; Lilley, Kathryn S.

    2014-01-01

    The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. PMID:25122472

  9. Quantification of the environmental impact of different dietary protein choices.

    PubMed

    Reijnders, Lucas; Soret, Sam

    2003-09-01

    Quantitative environmental evaluations of meat, fresh vegetables, and processed protein based on soybeans suggest that the environmental burden of vegetarian foods is usually relatively low when production and processing are considered. The environmental comparison of cheese varieties made from cow milk and directly from lupine and the evaluation of energy inputs in fish protein and vegetable protein also suggest an environmental advantage for vegetarian food. In the evaluation of processed protein food based on soybeans and meat protein, a variety of environmental impacts associated with primary production and processing are a factor 4.4-> 100 to the disadvantage of meat. The comparison of cheese varieties gives differences in specific environmental impacts ranging between a factor 5 and 21. And energy use for fish protein may be up to a factor 14 more than for protein of vegetable origin. Assessment suggests that on average the complete life cycle environmental impact of nonvegetarian meals may be roughly a factor 1.5-2 higher than the effect of vegetarian meals in which meat has been replaced by vegetable protein. Although on average vegetarian diets may well have an environmental advantage, exceptions may also occur. Long-distance air transport, deep-freezing, and some horticultural practices may lead to environmental burdens for vegetarian foods exceeding those for locally produced organic meat. PMID:12936964

  10. A six-plex proteome quantification strategy reveals the dynamics of protein turnover.

    PubMed

    Wang, Fangjun; Cheng, Kai; Wei, Xiaoluan; Qin, Hongqiang; Chen, Rui; Liu, Jing; Zou, Hanfa

    2013-01-01

    MS1 full scan based quantification is one of the most popular approaches for large-scale proteome quantification. Typically only three different samples can be differentially labeled and quantified in a single experiment. Here we present a two stages stable isotope labeling strategy which allows six different protein samples (six-plex) to be reliably labeled and simultaneously quantified at MS1 level. Briefly in the first stage, isotope lysine-d0 (K0) and lysine-d4 (K4) are in vivo incorporated into different protein samples during cell culture. Then in the second stage, three of K0 and K4 labeled protein samples are digested by lysine C and in vitro labeled with light (2CH3), medium (2CD2H), and heavy (2(13)CD3) dimethyl groups, respectively. We demonstrated that this six-plex isotope labeling strategy could successfully investigate the dynamics of protein turnover in a high throughput manner. PMID:23661174

  11. Quantification of photosensitized singlet oxygen production by a fluorescent protein.

    PubMed

    Ragàs, Xavier; Cooper, Laurie P; White, John H; Nonell, Santi; Flors, Cristina

    2011-01-17

    Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore-assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen ((1)O(2)). TagRFP is able to photosensitize (1)O(2) with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of (1)O(2) production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI. PMID:21226197

  12. Quantification of intact covalently metal labeled proteins using ESI-MS/MS.

    PubMed

    Benda, David; Schwarz, Gunnar; Beck, Sebastian; Linscheid, Michael W

    2014-01-01

    Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.

  13. Visualization and Quantification of Cardiac Mitochondrial Protein Clusters with STED Microscopy

    PubMed Central

    Singh, Harpreet; Lu, Rong; Rodríguez, Pedro Felipe Gardeazábal; Wu, Yong; Bopassa, Jean Chrisostome; Stefani, Enrico; Toro, Ligia

    2011-01-01

    The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~20 nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; specifically, cytochrome c oxidase subunit 2 is distributed in clusters of ~20 and ~28 nm; whereas the voltage dependent anion channel 1 displays four size distributions of ~22, ~33, ~55 and ~83 nm. PMID:21982778

  14. Validation of a fast and accurate chromatographic method for detailed quantification of vitamin E in green leafy vegetables.

    PubMed

    Cruz, Rebeca; Casal, Susana

    2013-11-15

    Vitamin E analysis in green vegetables is performed by an array of different methods, making it difficult to compare published data or choosing the adequate one for a particular sample. Aiming to achieve a consistent method with wide applicability, the current study reports the development and validation of a fast micro-method for quantification of vitamin E in green leafy vegetables. The methodology uses solid-liquid extraction based on the Folch method, with tocol as internal standard, and normal-phase HPLC with fluorescence detection. A large linear working range was confirmed, being highly reproducible, with inter-day precisions below 5% (RSD). Method sensitivity was established (below 0.02 μg/g fresh weight), and accuracy was assessed by recovery tests (>96%). The method was tested in different green leafy vegetables, evidencing diverse tocochromanol profiles, with variable ratios and amounts of α- and γ-tocopherol, and other minor compounds. The methodology is adequate for routine analyses, with a reduced chromatographic run (<7 min) and organic solvent consumption, and requires only standard chromatographic equipment available in most laboratories.

  15. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

    PubMed Central

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments. PMID:26302947

  16. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.

    PubMed

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

  17. Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

    SciTech Connect

    Shi, Tujin; Qian, Weijun

    2013-02-01

    Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

  18. An Overview of Practical Applications of Protein Disorder Prediction and Drive for Faster, More Accurate Predictions

    PubMed Central

    Deng, Xin; Gumm, Jordan; Karki, Suman; Eickholt, Jesse; Cheng, Jianlin

    2015-01-01

    Protein disordered regions are segments of a protein chain that do not adopt a stable structure. Thus far, a variety of protein disorder prediction methods have been developed and have been widely used, not only in traditional bioinformatics domains, including protein structure prediction, protein structure determination and function annotation, but also in many other biomedical fields. The relationship between intrinsically-disordered proteins and some human diseases has played a significant role in disorder prediction in disease identification and epidemiological investigations. Disordered proteins can also serve as potential targets for drug discovery with an emphasis on the disordered-to-ordered transition in the disordered binding regions, and this has led to substantial research in drug discovery or design based on protein disordered region prediction. Furthermore, protein disorder prediction has also been applied to healthcare by predicting the disease risk of mutations in patients and studying the mechanistic basis of diseases. As the applications of disorder prediction increase, so too does the need to make quick and accurate predictions. To fill this need, we also present a new approach to predict protein residue disorder using wide sequence windows that is applicable on the genomic scale. PMID:26198229

  19. CoMOGrad and PHOG: From Computer Vision to Fast and Accurate Protein Tertiary Structure Retrieval

    PubMed Central

    Karim, Rezaul; Aziz, Mohd. Momin Al; Shatabda, Swakkhar; Rahman, M. Sohel; Mia, Md. Abul Kashem; Zaman, Farhana; Rakin, Salman

    2015-01-01

    The number of entries in a structural database of proteins is increasing day by day. Methods for retrieving protein tertiary structures from such a large database have turn out to be the key to comparative analysis of structures that plays an important role to understand proteins and their functions. In this paper, we present fast and accurate methods for the retrieval of proteins having tertiary structures similar to a query protein from a large database. Our proposed methods borrow ideas from the field of computer vision. The speed and accuracy of our methods come from the two newly introduced features- the co-occurrence matrix of the oriented gradient and pyramid histogram of oriented gradient- and the use of Euclidean distance as the distance measure. Experimental results clearly indicate the superiority of our approach in both running time and accuracy. Our method is readily available for use from this website: http://research.buet.ac.bd:8080/Comograd/. PMID:26293226

  20. Residue level quantification of protein stability in living cells.

    PubMed

    Monteith, William B; Pielak, Gary J

    2014-08-01

    The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions. We report the use of NMR-detected hydrogen-deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data (pH 7.6 and 37 °C) show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, we also show that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. We discuss our findings in terms of hard-core excluded volume effects, charge-charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.

  1. Quantification of individual proteins in silicone hydrogel contact lens deposits

    PubMed Central

    Zhao, Zhenjun; Zhu, Hua; Tilia, Daniel; Willcox, Mark D.P.

    2013-01-01

    Purpose The aim of this study was to quantify specific proteins deposited on daily wear silicone hydrogel lenses used in combination with multipurpose disinfecting solutions (MPDSs) by applying multiple-reaction-monitoring mass spectrometry (MRM-MS). Methods Balafilcon A or senofilcon A contact lenses used with different MPDSs on a daily wear schedule were collected. Each worn lens was extracted and then digested with trypsin. MRM-MS was applied to quantify the amounts of lysozyme, lactoferrin, lipocalin-1, proline-rich protein-4, and keratin-1 in the extracts. Results The amount of protein extracted from the contact lenses was affected by the individual wearers, lens material, and type of care system used. Higher amounts of proteins were extracted from lenses after wear when they were used with an MPDS containing polyhexamethylene biguanide (PHMB) and poloxamer 407 compared with MPDSs containing polyquaternium-1 (PQ-1)/alexidine dihydrochloride with Tetronic 904 or PQ-1/ PHMB with poloxamine and sulfobetaine (p<0.05). There was a correlation between the amount of lipocalin-1 or keratin-1 extracted from lenses and symptoms of ocular dryness. Conclusions The MRM-MS technique is a promising approach that could be used to reveal associations of individual proteins deposited on lenses with performance of contact lenses during wear. PMID:23441110

  2. Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection.

    PubMed

    Lutter, Petra; Parisod, Véronique; Weymuth, Hans

    2011-01-01

    The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes. PMID:21919337

  3. Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

    PubMed

    Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

  4. A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins.

    PubMed

    Batth, Tanveer S; Singh, Pragya; Ramakrishnan, Vikram R; Sousa, Mirta M L; Chan, Leanne Jade G; Tran, Huu M; Luning, Eric G; Pan, Eva H Y; Vuu, Khanh M; Keasling, Jay D; Adams, Paul D; Petzold, Christopher J

    2014-11-01

    Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.

  5. Mass Spectrometry-based Workflow for Accurate Quantification of Escherichia coli Enzymes: How Proteomics Can Play a Key Role in Metabolic Engineering*

    PubMed Central

    Trauchessec, Mathieu; Jaquinod, Michel; Bonvalot, Aline; Brun, Virginie; Bruley, Christophe; Ropers, Delphine; de Jong, Hidde; Garin, Jérôme; Bestel-Corre, Gwenaëlle; Ferro, Myriam

    2014-01-01

    Metabolic engineering aims to design high performance microbial strains producing compounds of interest. This requires systems-level understanding; genome-scale models have therefore been developed to predict metabolic fluxes. However, multi-omics data including genomics, transcriptomics, fluxomics, and proteomics may be required to model the metabolism of potential cell factories. Recent technological advances to quantitative proteomics have made mass spectrometry-based quantitative assays an interesting alternative to more traditional immuno-affinity based approaches. This has improved specificity and multiplexing capabilities. In this study, we developed a quantification workflow to analyze enzymes involved in central metabolism in Escherichia coli (E. coli). This workflow combined full-length isotopically labeled standards with selected reaction monitoring analysis. First, full-length 15N labeled standards were produced and calibrated to ensure accurate measurements. Liquid chromatography conditions were then optimized for reproducibility and multiplexing capabilities over a single 30-min liquid chromatography-MS analysis. This workflow was used to accurately quantify 22 enzymes involved in E. coli central metabolism in a wild-type reference strain and two derived strains, optimized for higher NADPH production. In combination with measurements of metabolic fluxes, proteomics data can be used to assess different levels of regulation, in particular enzyme abundance and catalytic rate. This provides information that can be used to design specific strains used in biotechnology. In addition, accurate measurement of absolute enzyme concentrations is key to the development of predictive kinetic models in the context of metabolic engineering. PMID:24482123

  6. Stealth surface modification of surface-enhanced Raman scattering substrates for sensitive and accurate detection in protein solutions.

    PubMed

    Sun, Fang; Ella-Menye, Jean-Rene; Galvan, Daniel David; Bai, Tao; Hung, Hsiang-Chieh; Chou, Ying-Nien; Zhang, Peng; Jiang, Shaoyi; Yu, Qiuming

    2015-03-24

    Reliable surface-enhanced Raman scattering (SERS) based biosensing in complex media is impeded by nonspecific protein adsorptions. Because of the near-field effect of SERS, it is challenging to modify SERS-active substrates using conventional nonfouling materials without introducing interference from their SERS signals. Herein, we report a stealth surface modification strategy for sensitive, specific and accurate detection of fructose in protein solutions using SERS by forming a mixed self-assembled monolayer (SAM). The SAM consists of a short zwitterionic thiol, N,N-dimethyl-cysteamine-carboxybetaine (CBT), and a fructose probe 4-mercaptophenylboronic acid (4-MPBA). The specifically designed and synthesized CBT not only resists protein fouling effectively, but also has very weak Raman activity compared to 4-MPBA. Thus, the CBT SAM provides a stealth surface modification to SERS-active substrates. The surface compositions of mixed SAMs were investigated using X-ray photoelectron spectroscopy (XPS) and SERS, and their nonfouling properties were studied with a surface plasmon resonance (SPR) biosensor. The mixed SAM with a surface composition of 94% CBT demonstrated a very low bovine serum albumin (BSA) adsorption (∼3 ng/cm(2)), and moreover, only the 4-MPBA signal appeared in the SERS spectrum. With the use of this surface-modified SERS-active substrate, quantification of fructose over clinically relevant concentrations (0.01-1 mM) was achieved. Partial least-squares regression (PLS) analysis showed that the detection sensitivity and accuracy were maintained for the measurements in 1 mg/mL BSA solutions. This stealth surface modification strategy provides a novel route to introduce nonfouling property to SERS-active substrates for SERS biosensing in complex media.

  7. Direct and Absolute Quantification of over 1800 Yeast Proteins via Selected Reaction Monitoring*

    PubMed Central

    Lawless, Craig; Holman, Stephen W.; Brownridge, Philip; Lanthaler, Karin; Harman, Victoria M.; Watkins, Rachel; Hammond, Dean E.; Miller, Rebecca L.; Sims, Paul F. G.; Grant, Christopher M.; Eyers, Claire E.; Beynon, Robert J.

    2016-01-01

    Defining intracellular protein concentration is critical in molecular systems biology. Although strategies for determining relative protein changes are available, defining robust absolute values in copies per cell has proven significantly more challenging. Here we present a reference data set quantifying over 1800 Saccharomyces cerevisiae proteins by direct means using protein-specific stable-isotope labeled internal standards and selected reaction monitoring (SRM) mass spectrometry, far exceeding any previous study. This was achieved by careful design of over 100 QconCAT recombinant proteins as standards, defining 1167 proteins in terms of copies per cell and upper limits on a further 668, with robust CVs routinely less than 20%. The selected reaction monitoring-derived proteome is compared with existing quantitative data sets, highlighting the disparities between methodologies. Coupled with a quantification of the transcriptome by RNA-seq taken from the same cells, these data support revised estimates of several fundamental molecular parameters: a total protein count of ∼100 million molecules-per-cell, a median of ∼1000 proteins-per-transcript, and a linear model of protein translation explaining 70% of the variance in translation rate. This work contributes a “gold-standard” reference yeast proteome (including 532 values based on high quality, dual peptide quantification) that can be widely used in systems models and for other comparative studies. PMID:26750110

  8. AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.

    PubMed

    Zhang, Runxuan; Calixto, Cristiane P G; Tzioutziou, Nikoleta A; James, Allan B; Simpson, Craig G; Guo, Wenbin; Marquez, Yamile; Kalyna, Maria; Patro, Rob; Eyras, Eduardo; Barta, Andrea; Nimmo, Hugh G; Brown, John W S

    2015-10-01

    RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.

  9. Stable isotope dilution HILIC-MS/MS method for accurate quantification of glutamic acid, glutamine, pyroglutamic acid, GABA and theanine in mouse brain tissues.

    PubMed

    Inoue, Koichi; Miyazaki, Yasuto; Unno, Keiko; Min, Jun Zhe; Todoroki, Kenichiro; Toyo'oka, Toshimasa

    2016-01-01

    In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus.

  10. Quantification of Protein Expression Changes in the Aging Left Ventricle of Rattus norvegicus

    PubMed Central

    Grant, Jennifer E.; Bradshaw, Amy D.; Schwacke, John H.; Baicu, Catalin F.; Zile, Michael R.; Schey, Kevin L.

    2009-01-01

    As the heart ages, electrophysiological and biochemical changes can occur, and the ventricle in many cases loses distensibility, impairing diastolic function. How the proteomic signature of the aged ventricle is unique in comparison to young hearts is still under active investigation. We have undertaken a quantitative proteomics study of aging left ventricles (LVs) utilizing the isobaric Tagging for Relative and Absolute Quantification (iTRAQ) methodology. Differential protein expression was observed for 117 proteins including proteins involved in cell signaling, the immune response, structural proteins, and proteins mediating responses to oxidative stress. For many of these proteins, this is the first report of an association with the aged myocardium. Additionally, two proteins of unknown function were identified. This work serves as the basis for making future comparisons of the aged left ventricle proteome to that of left ventricles obtained from other models of disease and heart failure. PMID:19603826

  11. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    PubMed Central

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  12. SIFTER search: a web server for accurate phylogeny-based protein function prediction.

    PubMed

    Sahraeian, Sayed M; Luo, Kevin R; Brenner, Steven E

    2015-07-01

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access to precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. The SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded.

  13. Accurate quantification of two key time points used in the determination of hydroxyl polyaluminum species by ferron timed spectrophotometry.

    PubMed

    Zhang, Jing; Yong, Xiaojing; Zhao, Dongyan; Shi, Qiuyi

    2015-01-01

    The content of mononuclear Al (Ala%) changed with its determination time (ta) under different dosages of Ferron (7-iodo-8-hydroxyquinoline-5-sulfonic acid, [Ferron]), and the change of Ala% with [Ferron] at different ta was systematically investigated for the first time. Thus, the most appropriate ta was found with the optimal [Ferron]. Also, the judgment of the platform (flat or level portion) of the complete reaction on the absorption-time curve determined in the hydroxyl polyaluminum solution by Ferron timed spectrophotometry (Ferron assay) was first digitized. The time point (tb) of complete reaction between the medium polyaluminum (Alb) and Ferron reagent depended on the reaction extent, and time could not be used only to judge. Thus, the tb was accurately determined and reduced to half of original, which improved the experiment efficiency significantly. The Ferron assay was completely optimized.

  14. Real-Time Visualization and Quantification of Contractile Ring Proteins in Single Living Cells.

    PubMed

    Davidson, Reshma; Liu, Yajun; Gerien, Kenneth S; Wu, Jian-Qiu

    2016-01-01

    Single-cell microscopy provides a powerful tool to visualize cellular and subcellular processes in wild-type and mutant cells by observing fluorescently tagged proteins. Here, we describe three simple methods to visualize fission yeast cells: gelatin slides, coverslip-bottom dishes, and tetrad fluorescence microscopy. These imaging methods and data analysis using free software make it possible to quantify protein localization, dynamics, and concentration with high spatial and temporal resolution. In fission yeast, the actomyosin contractile ring is essential for cytokinesis. We use the visualization and quantification of contractile ring proteins as an example to demonstrate how to use these methods.

  15. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion

    SciTech Connect

    Shi, Tujin; Sun, Xuefei; Gao, Yuqian; Fillmore, Thomas L.; Schepmoes, Athena A.; Zhao, Rui; He, Jintang; Moore, Ronald J.; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Liu, Alvin Y.; Smith, Richard D.; Tang, Keqi; Camp, David G.; Qian, Weijun

    2013-07-05

    We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM) for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion and the multiplexing potential of this technique. Limits of quantification (LOQs) at low ng/mL levels with a median CV of ~12% were achieved for proteins spiked into human female serum using as little as 2 µL serum. PRISM-SRM provided up to ~1000-fold improvement in the LOQ when compared to conventional SRM measurements. Multiplexing capability of PRISM-SRM was also evaluated by two sets of serum samples with 6 and 21 target peptides spiked at the low attomole/µL levels. The results from SRM measurements for pooled or post-concatenated samples were comparable to those obtained from individual peptide fractions in terms of signal-to-noise ratios and SRM peak area ratios of light to heavy peptides. PRISM-SRM was applied to measure several ng/mL-level endogenous plasma proteins, including prostate-specific antigen, in clinical patient sera where correlation coefficients > 0.99 were observed between the results from PRISM-SRM and ELISA assays. Our results demonstrate that PRISM-SRM can be successfully used for quantification of low-abundance endogenous proteins in highly complex samples. Moderate throughput (50 samples/week) can be achieved by applying the post-concatenation or fraction multiplexing strategies. We anticipate broad applications for targeted PRISM

  16. Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue.

    PubMed

    Amisten, Stefan

    2016-01-01

    G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue. PMID:26928540

  17. Interference of salts used on aqueous two-phase systems on the quantification of total proteins.

    PubMed

    Golunski, Simone Maria; Sala, Luisa; Silva, Marceli Fernandes; Dallago, Rogério Marcos; Mulinari, Jéssica; Mossi, Altemir José; Brandelli, Adriano; Kalil, Susana Juliano; Di Luccio, Marco; Treichel, Helen

    2016-02-01

    In this study the interference of potassium phosphate, sodium citrate, sodium chloride and sodium nitrate salts on protein quantification by Bradford's method was assessed. Potassium phosphate and sodium citrate salts are commonly used in aqueous two-phase systems for enzyme purification. Results showed that the presence of potassium phosphate and sodium citrate salts increase the absorbance of the samples, when compared with the samples without any salt. The increase in absorptivity of the solution induces errors on protein quantification, which are propagated to the calculations of specific enzyme activity and consequently on purification factor. The presence of sodium chloride and sodium nitrate practically did not affect the absorbance of inulinase, probably the metals present in the enzyme extract did not interact with the added salts.

  18. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  19. Interference of salts used on aqueous two-phase systems on the quantification of total proteins.

    PubMed

    Golunski, Simone Maria; Sala, Luisa; Silva, Marceli Fernandes; Dallago, Rogério Marcos; Mulinari, Jéssica; Mossi, Altemir José; Brandelli, Adriano; Kalil, Susana Juliano; Di Luccio, Marco; Treichel, Helen

    2016-02-01

    In this study the interference of potassium phosphate, sodium citrate, sodium chloride and sodium nitrate salts on protein quantification by Bradford's method was assessed. Potassium phosphate and sodium citrate salts are commonly used in aqueous two-phase systems for enzyme purification. Results showed that the presence of potassium phosphate and sodium citrate salts increase the absorbance of the samples, when compared with the samples without any salt. The increase in absorptivity of the solution induces errors on protein quantification, which are propagated to the calculations of specific enzyme activity and consequently on purification factor. The presence of sodium chloride and sodium nitrate practically did not affect the absorbance of inulinase, probably the metals present in the enzyme extract did not interact with the added salts. PMID:26616454

  20. Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR.

    PubMed

    Thompson, Andrew R; Binder, Benjamin P; McCaffrey, Jesse E; Svensson, Bengt; Thomas, David D

    2015-01-01

    While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the label's stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination. PMID:26477249

  1. Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis.

    PubMed

    Liu, Hongcheng; Ponniah, Gomathinayagam; Neill, Alyssa; Patel, Rekha; Andrien, Bruce

    2013-12-17

    Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.

  2. Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum

    SciTech Connect

    Shi, Tujin; Fillmore, Thomas L.; Sun, Xuefei; Zhao, Rui; Schepmoes, Athena A.; Hossain, Mahmud; Xie, Fang; Wu, Si; Kim, Jong Seo; Jones, Nathaniel J.; Moore, Ronald J.; Pasa-Tolic, Ljiljana; Kagan, Jacob; Rodland, Karin D.; Liu, Tao; Tang, Keqi; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2012-09-18

    The detection and quantification of proteins at sub-ng/mL concentrations in plasma/serum is of critical importance for candidate biomarker verification. Sensitive detection of serum proteins has typically been achieved by immunoassays; however, de novo development of antibodies is associated with high cost and long lead time. To address this challenge, we developed an antibody-free strategy, termed high-pressure high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving accurate detection of proteins at ~50 pg/mL level in plasma/serum using selected reaction monitoring (SRM). This strategy is based on high resolution reversed phase liquid chromatographic separations for analyte enrichment along with intelligent selection of target fractions via on-line SRM monitoring of internal standards and fraction multiplexing prior to SRM quantification. Our work represents a major technological advance for achieving pg/mL level of serum protein quantification without specific affinity reagents and holds great promise for broad applications in biomarker verification and systems biology studies.

  3. Assessment of RT-qPCR normalization strategies for accurate quantification of extracellular microRNAs in murine serum.

    PubMed

    Roberts, Thomas C; Coenen-Stass, Anna M L; Wood, Matthew J A

    2014-01-01

    Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ∼2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide.

  4. Mass Spectrometry-Based Proteomics for Relative Protein Quantification and Biomarker Identification in Primary Human Hepatocytes.

    PubMed

    Dietz, Lisa; Sickmann, Albert

    2015-01-01

    Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The two-dimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results.

  5. An Accurate Method for Prediction of Protein-Ligand Binding Site on Protein Surface Using SVM and Statistical Depth Function

    PubMed Central

    Wang, Kui; Gao, Jianzhao; Shen, Shiyi; Tuszynski, Jack A.; Ruan, Jishou

    2013-01-01

    Since proteins carry out their functions through interactions with other molecules, accurately identifying the protein-ligand binding site plays an important role in protein functional annotation and rational drug discovery. In the past two decades, a lot of algorithms were present to predict the protein-ligand binding site. In this paper, we introduce statistical depth function to define negative samples and propose an SVM-based method which integrates sequence and structural information to predict binding site. The results show that the present method performs better than the existent ones. The accuracy, sensitivity, and specificity on training set are 77.55%, 56.15%, and 87.96%, respectively; on the independent test set, the accuracy, sensitivity, and specificity are 80.36%, 53.53%, and 92.38%, respectively. PMID:24195070

  6. Accurate refinement of docked protein complexes using evolutionary information and deep learning.

    PubMed

    Akbal-Delibas, Bahar; Farhoodi, Roshanak; Pomplun, Marc; Haspel, Nurit

    2016-06-01

    One of the major challenges for protein docking methods is to accurately discriminate native-like structures from false positives. Docking methods are often inaccurate and the results have to be refined and re-ranked to obtain native-like complexes and remove outliers. In a previous work, we introduced AccuRefiner, a machine learning based tool for refining protein-protein complexes. Given a docked complex, the refinement tool produces a small set of refined versions of the input complex, with lower root-mean-square-deviation (RMSD) of atomic positions with respect to the native structure. The method employs a unique ranking tool that accurately predicts the RMSD of docked complexes with respect to the native structure. In this work, we use a deep learning network with a similar set of features and five layers. We show that a properly trained deep learning network can accurately predict the RMSD of a docked complex with 1.40 Å error margin on average, by approximating the complex relationship between a wide set of scoring function terms and the RMSD of a docked structure. The network was trained on 35000 unbound docking complexes generated by RosettaDock. We tested our method on 25 different putative docked complexes produced also by RosettaDock for five proteins that were not included in the training data. The results demonstrate that the high accuracy of the ranking tool enables AccuRefiner to consistently choose the refinement candidates with lower RMSD values compared to the coarsely docked input structures. PMID:26846813

  7. Accurate refinement of docked protein complexes using evolutionary information and deep learning.

    PubMed

    Akbal-Delibas, Bahar; Farhoodi, Roshanak; Pomplun, Marc; Haspel, Nurit

    2016-06-01

    One of the major challenges for protein docking methods is to accurately discriminate native-like structures from false positives. Docking methods are often inaccurate and the results have to be refined and re-ranked to obtain native-like complexes and remove outliers. In a previous work, we introduced AccuRefiner, a machine learning based tool for refining protein-protein complexes. Given a docked complex, the refinement tool produces a small set of refined versions of the input complex, with lower root-mean-square-deviation (RMSD) of atomic positions with respect to the native structure. The method employs a unique ranking tool that accurately predicts the RMSD of docked complexes with respect to the native structure. In this work, we use a deep learning network with a similar set of features and five layers. We show that a properly trained deep learning network can accurately predict the RMSD of a docked complex with 1.40 Å error margin on average, by approximating the complex relationship between a wide set of scoring function terms and the RMSD of a docked structure. The network was trained on 35000 unbound docking complexes generated by RosettaDock. We tested our method on 25 different putative docked complexes produced also by RosettaDock for five proteins that were not included in the training data. The results demonstrate that the high accuracy of the ranking tool enables AccuRefiner to consistently choose the refinement candidates with lower RMSD values compared to the coarsely docked input structures.

  8. Accurate Quantification of Selenoprotein P (SEPP1) in Plasma Using Isotopically Enriched Seleno-peptides and Species-Specific Isotope Dilution with HPLC Coupled to ICP-MS/MS.

    PubMed

    Deitrich, Christian L; Cuello-Nuñez, Susana; Kmiotek, Diana; Torma, Frank Attila; Del Castillo Busto, Maria Estela; Fisicaro, Paola; Goenaga-Infante, Heidi

    2016-06-21

    A novel strategy for the absolute quantification of selenium (Se) included in selenoprotein P (SEPP1), an important biomarker for human nutrition and disease, including diabetes and cancer, is presented here for the first time. It is based on the use of species-specific double isotope dilution mass spectrometry (SSIDA) in combination with HPLC-ICP-MS/MS for the determination of protein bound Se down to the peptide level in a complex plasma matrix with a total content of Se of 105.5 μg kg(-1). The method enabled the selective Se speciation analysis of human plasma samples without the need of extensive cleanup or preconcentration steps as required for traditional protein mass spectrometric approaches. To assess the method accuracy, two plasma reference materials, namely, BCR-637 and SRM1950, for which literature data and a reference value for SEPP1 have been reported, were analyzed using complementary hyphenated methods and the species-specific approach developed in this work. The Se mass fractions obtained via the isotopic ratios (78)Se/(76)Se and (82)Se/(76)Se for each of the Se-peptides, namely, ENLPSLCSUQGLR (ENL) and AEENITESCQUR (AEE) (where U is SeCys), were found to agree within 2.4%. A relative expanded combined uncertainty (k = 2) of 5.4% was achieved for a Se (as SEPP1) mass fraction of approximately 60 μg kg(-1). This work represents a systematic approach to the accurate quantitation of plasma SEPP1 at clinical levels using SSIDA quantification. Such methodology will be invaluable for the certification of reference materials and the provision of reference values to clinical measurements and clinical trials. PMID:27108743

  9. Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives.

    PubMed

    de Fátima MenegociEugênio, Patrícia; Assunção, Nilson Antonio; Sciandra, Francesca; Aquino, Adriano; Brancaccio, Andrea; Carrilho, Emanuel

    2016-01-01

    One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre-purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R-250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.

  10. Rapid detection and quantification of specific proteins by immunodepletion and microfluidic separation

    PubMed Central

    Azadi, Glareh; Gustafson, Eric; Wessel, Gary M.; Tripathi, Anubhav

    2012-01-01

    Conventional immunoblotting techniques are labor intensive, time consuming and rely on the elution of target protein after depletion. Here we describe a new method for detection and quantification of proteins, independent of washing and elution. In this method, the target protein is first captured by immunodepletion with antibody coated microbeads. In the second step, both the supernatant after immunodepletion and the untreated protein sample are directly analyzed by microfluidic electrophoresis without further processing. Subsequently, the detection and quantification are performed comparing the electropherograms of these two samples. This method was tested using an Escherichia coli lysate with a FLAG-tagged protein and anti-FLAG magnetic beads. An incubation of as little as one minute was sufficient for detectable depletion (66%) by microchip electrophoresis. Longer incubation (up to 60 minutes) resulted in more depletion of the target band (82%). Our results show that only 19% of the target is recovered after elution from the beads. By eliminating multiple wash and elution steps, our method is faster, less labor intensive, and highly reproducible. Even in case of non-specific binding at low concentrations, the target protein can be easily identified. This work highlights the advantages of integrating immunodepletion techniques on a microfluidic platform. PMID:22539461

  11. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics. PMID:27314233

  12. A NaBH₄ Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass.

    PubMed

    Mok, Yiu Ki; Arantes, Valdeir; Saddler, Jack N

    2015-07-01

    Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination. PMID:25987134

  13. Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

    PubMed Central

    Smits, Arne H.; Lindeboom, Rik G.H.; Perino, Matteo; van Heeringen, Simon J.; Veenstra, Gert Jan C.; Vermeulen, Michiel

    2014-01-01

    While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs. PMID:25056316

  14. Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis *

    PubMed Central

    Maaβ, Sandra; Wachlin, Gerhild; Bernhardt, Jörg; Eymann, Christine; Fromion, Vincent; Riedel, Katharina; Becher, Dörte; Hecker, Michael

    2014-01-01

    Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. PMID:24878497

  15. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    PubMed

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein.

  16. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    PubMed

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein. PMID:26795634

  17. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC.

  18. Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

    PubMed Central

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-01-01

    Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA–protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA–protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA–protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA–protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1–DNA and Top2a–DNA adducts in human cells, and gyrase–DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  19. A multi-objective optimization approach accurately resolves protein domain architectures

    PubMed Central

    Bernardes, J.S.; Vieira, F.R.J.; Zaverucha, G.; Carbone, A.

    2016-01-01

    Motivation: Given a protein sequence and a number of potential domains matching it, what are the domain content and the most likely domain architecture for the sequence? This problem is of fundamental importance in protein annotation, constituting one of the main steps of all predictive annotation strategies. On the other hand, when potential domains are several and in conflict because of overlapping domain boundaries, finding a solution for the problem might become difficult. An accurate prediction of the domain architecture of a multi-domain protein provides important information for function prediction, comparative genomics and molecular evolution. Results: We developed DAMA (Domain Annotation by a Multi-objective Approach), a novel approach that identifies architectures through a multi-objective optimization algorithm combining scores of domain matches, previously observed multi-domain co-occurrence and domain overlapping. DAMA has been validated on a known benchmark dataset based on CATH structural domain assignments and on the set of Plasmodium falciparum proteins. When compared with existing tools on both datasets, it outperforms all of them. Availability and implementation: DAMA software is implemented in C++ and the source code can be found at http://www.lcqb.upmc.fr/DAMA. Contact: juliana.silva_bernardes@upmc.fr or alessandra.carbone@lip6.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26458889

  20. SIFTER search: a web server for accurate phylogeny-based protein function prediction

    DOE PAGES

    Sahraeian, Sayed M.; Luo, Kevin R.; Brenner, Steven E.

    2015-05-15

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access tomore » precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. Lastly, the SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded.« less

  1. SIFTER search: a web server for accurate phylogeny-based protein function prediction.

    PubMed

    Sahraeian, Sayed M; Luo, Kevin R; Brenner, Steven E

    2015-07-01

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access to precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. The SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded. PMID:25979264

  2. SIFTER search: a web server for accurate phylogeny-based protein function prediction

    SciTech Connect

    Sahraeian, Sayed M.; Luo, Kevin R.; Brenner, Steven E.

    2015-05-15

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access to precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. Lastly, the SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded.

  3. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    SciTech Connect

    Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  4. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

    PubMed

    Malik, Afshan N; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  5. Rapid calculation of accurate atomic charges for proteins via the electronegativity equalization method.

    PubMed

    Ionescu, Crina-Maria; Geidl, Stanislav; Svobodová Vařeková, Radka; Koča, Jaroslav

    2013-10-28

    We focused on the parametrization and evaluation of empirical models for fast and accurate calculation of conformationally dependent atomic charges in proteins. The models were based on the electronegativity equalization method (EEM), and the parametrization procedure was tailored to proteins. We used large protein fragments as reference structures and fitted the EEM model parameters using atomic charges computed by three population analyses (Mulliken, Natural, iterative Hirshfeld), at the Hartree-Fock level with two basis sets (6-31G*, 6-31G**) and in two environments (gas phase, implicit solvation). We parametrized and successfully validated 24 EEM models. When tested on insulin and ubiquitin, all models reproduced quantum mechanics level charges well and were consistent with respect to population analysis and basis set. Specifically, the models showed on average a correlation of 0.961, RMSD 0.097 e, and average absolute error per atom 0.072 e. The EEM models can be used with the freely available EEM implementation EEM_SOLVER.

  6. Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra

    PubMed Central

    Xiao, Kaijie; Yu, Fan; Fang, Houqin; Xue, Bingbing; Liu, Yan; Tian, Zhixin

    2015-01-01

    It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available. PMID:26439836

  7. Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis.

    PubMed

    Zhu, Lijuan; Wang, Zhikai; Wang, Wenwen; Wang, Chunli; Hua, Shasha; Su, Zeqi; Brako, Larry; Garcia-Barrio, Minerva; Ye, Mingliang; Wei, Xuan; Zou, Hanfa; Ding, Xia; Liu, Lifang; Liu, Xing; Yao, Xuebiao

    2015-11-01

    Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.

  8. Electrostatics of proteins in dielectric solvent continua. I. An accurate and efficient reaction field description

    SciTech Connect

    Bauer, Sebastian; Mathias, Gerald; Tavan, Paul

    2014-03-14

    We present a reaction field (RF) method which accurately solves the Poisson equation for proteins embedded in dielectric solvent continua at a computational effort comparable to that of an electrostatics calculation with polarizable molecular mechanics (MM) force fields. The method combines an approach originally suggested by Egwolf and Tavan [J. Chem. Phys. 118, 2039 (2003)] with concepts generalizing the Born solution [Z. Phys. 1, 45 (1920)] for a solvated ion. First, we derive an exact representation according to which the sources of the RF potential and energy are inducible atomic anti-polarization densities and atomic shielding charge distributions. Modeling these atomic densities by Gaussians leads to an approximate representation. Here, the strengths of the Gaussian shielding charge distributions are directly given in terms of the static partial charges as defined, e.g., by standard MM force fields for the various atom types, whereas the strengths of the Gaussian anti-polarization densities are calculated by a self-consistency iteration. The atomic volumes are also described by Gaussians. To account for covalently overlapping atoms, their effective volumes are calculated by another self-consistency procedure, which guarantees that the dielectric function ε(r) is close to one everywhere inside the protein. The Gaussian widths σ{sub i} of the atoms i are parameters of the RF approximation. The remarkable accuracy of the method is demonstrated by comparison with Kirkwood's analytical solution for a spherical protein [J. Chem. Phys. 2, 351 (1934)] and with computationally expensive grid-based numerical solutions for simple model systems in dielectric continua including a di-peptide (Ac-Ala-NHMe) as modeled by a standard MM force field. The latter example shows how weakly the RF conformational free energy landscape depends on the parameters σ{sub i}. A summarizing discussion highlights the achievements of the new theory and of its approximate solution

  9. Electrostatics of proteins in dielectric solvent continua. I. An accurate and efficient reaction field description.

    PubMed

    Bauer, Sebastian; Mathias, Gerald; Tavan, Paul

    2014-03-14

    We present a reaction field (RF) method which accurately solves the Poisson equation for proteins embedded in dielectric solvent continua at a computational effort comparable to that of an electrostatics calculation with polarizable molecular mechanics (MM) force fields. The method combines an approach originally suggested by Egwolf and Tavan [J. Chem. Phys. 118, 2039 (2003)] with concepts generalizing the Born solution [Z. Phys. 1, 45 (1920)] for a solvated ion. First, we derive an exact representation according to which the sources of the RF potential and energy are inducible atomic anti-polarization densities and atomic shielding charge distributions. Modeling these atomic densities by Gaussians leads to an approximate representation. Here, the strengths of the Gaussian shielding charge distributions are directly given in terms of the static partial charges as defined, e.g., by standard MM force fields for the various atom types, whereas the strengths of the Gaussian anti-polarization densities are calculated by a self-consistency iteration. The atomic volumes are also described by Gaussians. To account for covalently overlapping atoms, their effective volumes are calculated by another self-consistency procedure, which guarantees that the dielectric function ε(r) is close to one everywhere inside the protein. The Gaussian widths σ(i) of the atoms i are parameters of the RF approximation. The remarkable accuracy of the method is demonstrated by comparison with Kirkwood's analytical solution for a spherical protein [J. Chem. Phys. 2, 351 (1934)] and with computationally expensive grid-based numerical solutions for simple model systems in dielectric continua including a di-peptide (Ac-Ala-NHMe) as modeled by a standard MM force field. The latter example shows how weakly the RF conformational free energy landscape depends on the parameters σ(i). A summarizing discussion highlights the achievements of the new theory and of its approximate solution particularly by

  10. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  11. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids.

    PubMed

    Hesse, Almut; Weller, Michael G

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  12. FAMSA: Fast and accurate multiple sequence alignment of huge protein families

    PubMed Central

    Deorowicz, Sebastian; Debudaj-Grabysz, Agnieszka; Gudyś, Adam

    2016-01-01

    Rapid development of modern sequencing platforms has contributed to the unprecedented growth of protein families databases. The abundance of sets containing hundreds of thousands of sequences is a formidable challenge for multiple sequence alignment algorithms. The article introduces FAMSA, a new progressive algorithm designed for fast and accurate alignment of thousands of protein sequences. Its features include the utilization of the longest common subsequence measure for determining pairwise similarities, a novel method of evaluating gap costs, and a new iterative refinement scheme. What matters is that its implementation is highly optimized and parallelized to make the most of modern computer platforms. Thanks to the above, quality indicators, i.e. sum-of-pairs and total-column scores, show FAMSA to be superior to competing algorithms, such as Clustal Omega or MAFFT for datasets exceeding a few thousand sequences. Quality does not compromise on time or memory requirements, which are an order of magnitude lower than those in the existing solutions. For example, a family of 415519 sequences was analyzed in less than two hours and required no more than 8 GB of RAM. FAMSA is available for free at http://sun.aei.polsl.pl/REFRESH/famsa. PMID:27670777

  13. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    PubMed

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

  14. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    PubMed

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

  15. Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations.

    PubMed

    Porebski, Przemyslaw Jerzy; Cymborowski, Marcin; Pasenkiewicz-Gierula, Marta; Minor, Wladek

    2016-02-01

    Improvements in crystallographic hardware and software have allowed automated structure-solution pipelines to approach a near-`one-click' experience for the initial determination of macromolecular structures. However, in many cases the resulting initial model requires a laborious, iterative process of refinement and validation. A new method has been developed for the automatic modeling of side-chain conformations that takes advantage of rotamer-prediction methods in a crystallographic context. The algorithm, which is based on deterministic dead-end elimination (DEE) theory, uses new dense conformer libraries and a hybrid energy function derived from experimental data and prior information about rotamer frequencies to find the optimal conformation of each side chain. In contrast to existing methods, which incorporate the electron-density term into protein-modeling frameworks, the proposed algorithm is designed to take advantage of the highly discriminatory nature of electron-density maps. This method has been implemented in the program Fitmunk, which uses extensive conformational sampling. This improves the accuracy of the modeling and makes it a versatile tool for crystallographic model building, refinement and validation. Fitmunk was extensively tested on over 115 new structures, as well as a subset of 1100 structures from the PDB. It is demonstrated that the ability of Fitmunk to model more than 95% of side chains accurately is beneficial for improving the quality of crystallographic protein models, especially at medium and low resolutions. Fitmunk can be used for model validation of existing structures and as a tool to assess whether side chains are modeled optimally or could be better fitted into electron density. Fitmunk is available as a web service at http://kniahini.med.virginia.edu/fitmunk/server/ or at http://fitmunk.bitbucket.org/.

  16. Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations

    PubMed Central

    Porebski, Przemyslaw Jerzy; Cymborowski, Marcin; Pasenkiewicz-Gierula, Marta; Minor, Wladek

    2016-01-01

    Improvements in crystallographic hardware and software have allowed automated structure-solution pipelines to approach a near-‘one-click’ experience for the initial determination of macromolecular structures. However, in many cases the resulting initial model requires a laborious, iterative process of refinement and validation. A new method has been developed for the automatic modeling of side-chain conformations that takes advantage of rotamer-prediction methods in a crystallographic context. The algorithm, which is based on deterministic dead-end elimination (DEE) theory, uses new dense conformer libraries and a hybrid energy function derived from experimental data and prior information about rotamer frequencies to find the optimal conformation of each side chain. In contrast to existing methods, which incorporate the electron-density term into protein-modeling frameworks, the proposed algorithm is designed to take advantage of the highly discriminatory nature of electron-density maps. This method has been implemented in the program Fitmunk, which uses extensive conformational sampling. This improves the accuracy of the modeling and makes it a versatile tool for crystallographic model building, refinement and validation. Fitmunk was extensively tested on over 115 new structures, as well as a subset of 1100 structures from the PDB. It is demonstrated that the ability of Fitmunk to model more than 95% of side chains accurately is beneficial for improving the quality of crystallographic protein models, especially at medium and low resolutions. Fitmunk can be used for model validation of existing structures and as a tool to assess whether side chains are modeled optimally or could be better fitted into electron density. Fitmunk is available as a web service at http://kniahini.med.virginia.edu/fitmunk/server/ or at http://fitmunk.bitbucket.org/. PMID:26894674

  17. Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations.

    PubMed

    Porebski, Przemyslaw Jerzy; Cymborowski, Marcin; Pasenkiewicz-Gierula, Marta; Minor, Wladek

    2016-02-01

    Improvements in crystallographic hardware and software have allowed automated structure-solution pipelines to approach a near-`one-click' experience for the initial determination of macromolecular structures. However, in many cases the resulting initial model requires a laborious, iterative process of refinement and validation. A new method has been developed for the automatic modeling of side-chain conformations that takes advantage of rotamer-prediction methods in a crystallographic context. The algorithm, which is based on deterministic dead-end elimination (DEE) theory, uses new dense conformer libraries and a hybrid energy function derived from experimental data and prior information about rotamer frequencies to find the optimal conformation of each side chain. In contrast to existing methods, which incorporate the electron-density term into protein-modeling frameworks, the proposed algorithm is designed to take advantage of the highly discriminatory nature of electron-density maps. This method has been implemented in the program Fitmunk, which uses extensive conformational sampling. This improves the accuracy of the modeling and makes it a versatile tool for crystallographic model building, refinement and validation. Fitmunk was extensively tested on over 115 new structures, as well as a subset of 1100 structures from the PDB. It is demonstrated that the ability of Fitmunk to model more than 95% of side chains accurately is beneficial for improving the quality of crystallographic protein models, especially at medium and low resolutions. Fitmunk can be used for model validation of existing structures and as a tool to assess whether side chains are modeled optimally or could be better fitted into electron density. Fitmunk is available as a web service at http://kniahini.med.virginia.edu/fitmunk/server/ or at http://fitmunk.bitbucket.org/. PMID:26894674

  18. Nonspecific particle-based method with two-photon excitation detection for sensitive protein quantification and cell counting.

    PubMed

    Pihlasalo, Sari; Engbert, Anke; Martikkala, Eija; Ylander, Pilvi; Hänninen, Pekka; Härmä, Harri

    2013-03-01

    A novel easy-to-use homogeneous method utilizing two-photon excitation (TPX) for quantification of proteins or counting of eukaryotic cells in solution has been developed. This highly sensitive technique is based on the adsorption competition between the sample and fluorescently labeled protein to micrometer-sized carboxylate modified polystyrene particles and detection of two-photon excited fluorescence. The adsorption of the labeled protein to the particles was detected as a distinct fluorescence on individual microparticles. Analyte protein or eukaryotic cells interacted with particle surface and reduced the adsorption of labeled protein to the particles resulting in a decrease of the fluorescence. The optimizations of assay conditions were performed separately for protein quantification and cell counting, and the principle of the method was confirmed with the fluorescence microscopy imaging. The protein quantification assay allowed the determination of picogram quantities (1.2 μg/L) of protein, and the cell counting assay allowed three cells in the sample with an average variation of approximately 10% in the signal. The protein assay sensitivity was more than 500-fold improved from the common most sensitive commercial methods. Moreover, the dynamic range of the assay was broad, approximately 4 orders of magnitude. The cell assay has sensitivity comparable to the most sensitive commercial method. The developed method tolerates interfering agents such as neutral detergents found in cell lysate samples even at high concentrations. The method is experimentally fairly simple and allows the expansion for the use of the TPX technology.

  19. RAP: Accurate and Fast Motif Finding Based on Protein-Binding Microarray Data

    PubMed Central

    Orenstein, Yaron; Mick, Eran

    2013-01-01

    Abstract The novel high-throughput technology of protein-binding microarrays (PBMs) measures binding intensity of a transcription factor to thousands of DNA probe sequences. Several algorithms have been developed to extract binding-site motifs from these data. Such motifs are commonly represented by positional weight matrices. Previous studies have shown that the motifs produced by these algorithms are either accurate in predicting in vitro binding or similar to previously published motifs, but not both. In this work, we present a new simple algorithm to infer binding-site motifs from PBM data. It outperforms prior art both in predicting in vitro binding and in producing motifs similar to literature motifs. Our results challenge previous claims that motifs with lower information content are better models for transcription-factor binding specificity. Moreover, we tested the effect of motif length and side positions flanking the “core” motif in the binding site. We show that side positions have a significant effect and should not be removed, as commonly done. A large drop in the results quality of all methods is observed between in vitro and in vivo binding prediction. The software is available on acgt.cs.tau.ac.il/rap. PMID:23464877

  20. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  1. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition.

    PubMed

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-28

    Reengineering protein-protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of "second-site suppressors," where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein-protein interfaces. To extend this approach, it would be advantageous to be able to "transplant" existing engineered and experimentally validated specificity changes to other homologous protein-protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain-peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein-protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein-protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context. PMID:25313039

  2. Accurate Quantification of Ionospheric State Based on Comprehensive Radiative Transfer Modeling and Optimal Inversion of the OI 135.6-nm Emission

    NASA Astrophysics Data System (ADS)

    Qin, J.; Kamalabadi, F.; Makela, J. J.; Meier, R. J.

    2015-12-01

    Remote sensing of the nighttime OI 135.6-nm emission represents the primary means of quantifying the F-region ionospheric state from optical measurements. Despite its pervasive use for studying aeronomical processes, the interpretation of these emissions as a proxy for ionospheric state remains ambiguous in that the relative contributions of radiative recombination and mutual neutralization to the production and, especially, the effects of scattering and absorption on the transport of the 135.6-nm emissions have not been fully quantified. Moreover, an inversion algorithm, which is robust to varying ionospheric structures under different geophysical conditions, is yet to be developed for statistically optimal characterization of the ionospheric state. In this work, as part of the NASA ICON mission, we develop a comprehensive radiative transfer model from first principle to investigate the production and transport of the nighttime 135.6-nm emissions. The forward modeling investigation indicates that under certain conditions mutual neutralization can contribute up to ~38% to the 135.6-nm emissions. Moreover, resonant scattering and pure absorption can reduce the brightness observed in the limb direction by ~40% while enhancing the brightness in the nadir direction by ~25%. Further analysis shows that without properly addressing these effects in the inversion process, the peak electron density in the F-region ionosphere (NmF2) can be overestimated by up to ~24%. To address these issues, an inversion algorithm that properly accounts for the above-mentioned effects is proposed for accurate quantification of the ionospheric state using satellite measurements. The ill-posedness due to the intrinsic presence of noise in real data is coped with by incorporating proper regularizations that enforce either global smoothness or piecewise smoothness of the solution. Application to model-generated data with different signal-to-noise ratios show that the algorithm has achieved

  3. Accurate quantification of the mercapturic acids of acrylonitrile and its genotoxic metabolite cyanoethylene-epoxide in human urine by isotope-dilution LC-ESI/MS/MS.

    PubMed

    Schettgen, T; Bertram, J; Kraus, T

    2012-08-30

    Acrylonitrile is a highly important industrial chemical with a high production volume worldwide, especially in the plastics industry. It is classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC group 2B). During metabolism of acrylonitrile, the genotoxic metabolite cyanoethylene-epoxide is formed. The urinary mercapturic acids of acrylonitrile, namely N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA) and cyanoethylene-epoxide, namely N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (CHEMA) are specific biomarkers for the determination of individual internal exposure to acrylonitrile and its highly reactive metabolite. We have developed and validated a sensitive method for the accurate determination of CEMA and CHEMA in human urine with a multidimensional LC/MS/MS-method using deuterium-labelled analogues for both analytes as internal standards. Analytes were stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and determined by tandem mass spectrometry. The limit of quantification (LOQ) for CEMA and CHEMA was 1 μg/L urine and allowed to quantify the background exposure of the (smoking) general population. Precision within and between series for CHEMA ranged from 2.6 to 8.0% at four concentrations ranging from 8.3 to 86 μg/L urine, mean accuracy was between 94 and 100%. For CEMA, precision within and between series ranged from 2.4 to 14.5% at four concentrations ranging from 15.1 to 196 μg/L urine, mean accuracy was between 91 and 104%. We applied the method to spot urine samples of 83 subjects of the general population with no known occupational exposure to acrylonitrile. Median levels (range) for CEMA and CHEMA in urine samples of non-smokers (n=47) were 1.9 μg/L (<1-16.4 μg/L) and<1 μg/L (<1-3 μg/L), while in urine samples of smokers (n=36), median levels were 184 μg/L (2-907 μg/L) and 29.3 μg/L (<1-147 μg/L), respectively. Smokers showed a

  4. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC. PMID:23122071

  5. Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification.

    PubMed

    Zhi, Wenbo; Wang, Meiyao; She, Jin-Xiong

    2011-06-15

    The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrate the feasibility of using selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG), have good linear ranges, generally 10(3) to 10(4) . There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R(2) is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins within the range of 0.2-2 fmol, which is comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R(2)=0.928 and 0.851, respectively). However, the correlation between SRM/MRM and ELISA for CP is less desirable (R(2)=0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides being analyzed (R(2)=0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). The SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or LUMINEX are not available. PMID:21594933

  6. Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

    PubMed

    Chen, Chaoxiang; Zhang, Xiang; Zhang, Shuyue; Zhu, Shaobin; Xu, Jingyi; Zheng, Yan; Han, Jinyan; Zeng, Jin-Zhang; Yan, Xiaomei

    2015-12-15

    Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.

  7. Accurate and precise quantification of atmospheric nitrate in streams draining land of various uses by using triple oxygen isotopes as tracers

    NASA Astrophysics Data System (ADS)

    Tsunogai, Urumu; Miyauchi, Takanori; Ohyama, Takuya; Komatsu, Daisuke D.; Nakagawa, Fumiko; Obata, Yusuke; Sato, Keiichi; Ohizumi, Tsuyoshi

    2016-06-01

    Land use in a catchment area has significant impacts on nitrate eluted from the catchment, including atmospheric nitrate deposited onto the catchment area and remineralised nitrate produced within the catchment area. Although the stable isotopic compositions of nitrate eluted from a catchment can be a useful tracer to quantify the land use influences on the sources and behaviour of the nitrate, it is best to determine these for the remineralised portion of the nitrate separately from the unprocessed atmospheric nitrate to obtain a more accurate and precise quantification of the land use influences. In this study, we determined the spatial distribution and seasonal variation of stable isotopic compositions of nitrate for more than 30 streams within the same watershed, the Lake Biwa watershed in Japan, in order to use 17O excess (Δ17O) of nitrate as an additional tracer to quantify the mole fraction of atmospheric nitrate accurately and precisely. The stable isotopic compositions, including Δ17O of nitrate, in precipitation (wet deposition; n = 196) sampled at the Sado-seki monitoring station were also determined for 3 years. The deposited nitrate showed large 17O excesses similar to those already reported for midlatitudes: Δ17O values ranged from +18.6 to +32.4 ‰ with a 3-year average of +26.3 ‰. However, nitrate in each inflow stream showed small annual average Δ17O values ranging from +0.5 to +3.1 ‰, which corresponds to mole fractions of unprocessed atmospheric nitrate to total nitrate from (1.8 ± 0.3) to (11.8 ± 1.8) % respectively, with an average for all inflow streams of (5.1 ± 0.5) %. Although the annual average Δ17O values tended to be smaller in accordance with the increase in annual average stream nitrate concentration from 12.7 to 106.2 µmol L-1, the absolute concentrations of unprocessed atmospheric nitrate were almost stable at (2.3 ± 1.1) µmol L-1 irrespective of the changes in population density and land use in each catchment area

  8. Rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system.

    PubMed

    Kung, S H; Wang, Y C; Lin, C H; Kuo, R L; Liu, W T

    2000-11-01

    A genetically modified cell line (Vero-ICP10-EGFP) was constructed for detection of herpes simplex virus (HSV) by a simple, rapid and direct method. The cell line was developed by stable transfection of Vero cell with a plasmid encoding the green fluorescent protein (GFP) driven by the promoter of the HSV-2 ICP10 gene. As early as 6 h after infection with HSV, fluorescence-emitting cells can be observed under a fluorescence microscope. A single infected cell emitting fluorescence can be observed with soft agar overlay by inverted fluorescence microscopy. No induction of detectable fluorescence was seen following infections with human cytomegalovirus (HCMV), varicella zoster virus (VZV), coxsackievirus A16 and enterovirus 71. Analysis by flow cytometry also demonstrated that intensity of the triggered fluorescence is proportional to the titer of HSV inoculated. Taken together, this novel GFP reporter system could become a useful means for rapid detection and quantification of HSV in clinical specimens.

  9. Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice.

    PubMed

    Jakubowski, Hieronim

    2016-09-01

    Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2-20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.

  10. Detection and quantification of protein oxidation in sarcopenic models: a mass spectrometry study.

    PubMed

    Pasha, Sabah; Tveen Jensen, Karina; Pitt, Andrew R; Spickett, Corinne M

    2014-10-01

    Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies. PMID:26461380

  11. Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein.

    PubMed

    Sinawang, Prima Dewi; Rai, Varun; Ionescu, Rodica E; Marks, Robert S

    2016-03-15

    An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25 ng/mL with an ultrasensitive detection limit of 0.5 ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein.

  12. Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein.

    PubMed

    Sinawang, Prima Dewi; Rai, Varun; Ionescu, Rodica E; Marks, Robert S

    2016-03-15

    An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25 ng/mL with an ultrasensitive detection limit of 0.5 ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein. PMID:26433352

  13. Accurate and Rigorous Prediction of the Changes in Protein Free Energies in a Large-Scale Mutation Scan.

    PubMed

    Gapsys, Vytautas; Michielssens, Servaas; Seeliger, Daniel; de Groot, Bert L

    2016-06-20

    The prediction of mutation-induced free-energy changes in protein thermostability or protein-protein binding is of particular interest in the fields of protein design, biotechnology, and bioengineering. Herein, we achieve remarkable accuracy in a scan of 762 mutations estimating changes in protein thermostability based on the first principles of statistical mechanics. The remaining error in the free-energy estimates appears to be due to three sources in approximately equal parts, namely sampling, force-field inaccuracies, and experimental uncertainty. We propose a consensus force-field approach, which, together with an increased sampling time, leads to a free-energy prediction accuracy that matches those reached in experiments. This versatile approach enables accurate free-energy estimates for diverse proteins, including the prediction of changes in the melting temperature of the membrane protein neurotensin receptor 1. PMID:27122231

  14. Hybrid quadrupole-orbitrap mass spectrometry analysis with accurate-mass database and parallel reaction monitoring for high-throughput screening and quantification of multi-xenobiotics in honey.

    PubMed

    Li, Yi; Zhang, Jinzhen; Jin, Yue; Wang, Lin; Zhao, Wen; Zhang, Wenwen; Zhai, Lifei; Zhang, Yaping; Zhang, Yongxin; Zhou, Jinhui

    2016-01-15

    This study reports a rapid, automated screening and quantification method for the determination of multi-xenobiotic residues in honey using ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap) with a user-built accurate-mass database plus parallel reaction monitoring (PRM). The database contains multi-xenobiotic information including formulas, adduct types, theoretical exact mass and retention time, characteristic fragment ions, ion ratios, and mass accuracies. A simple sample preparation method was developed to reduce xenobiotic loss in the honey samples. The screening method was validated based on retention time deviation, mass accuracy via full scan-data-dependent MS/MS (full scan-ddMS2), multi-isotope ratio, characteristic ion ratio, sensitivity, and positive/negative switching performance between the spiked sample and corresponding standard solution. The quantification method based on the PRM mode is a promising new quantitative tool which we validated in terms of selectivity, linearity, recovery (accuracy), repeatability (precision), decision limit (CCα), detection capability (CCβ), matrix effects, and carry-over. The optimized methods proposed in this study enable the automated screening and quantification of 157 compounds in less than 15 min in honey. The results of this study, as they represent a convenient protocol for large-scale screening and quantification, also provide a research approach for analysis of various contaminants in other matrices.

  15. Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms.

    PubMed

    Peck, Amy R; Girondo, Melanie A; Liu, Chengbao; Kovatich, Albert J; Hooke, Jeffrey A; Shriver, Craig D; Hu, Hai; Mitchell, Edith P; Freydin, Boris; Hyslop, Terry; Chervoneva, Inna; Rui, Hallgeir

    2016-10-01

    Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan-Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0

  16. Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms

    PubMed Central

    Peck, Amy R; Girondo, Melanie A; Liu, Chengbao; Kovatich, Albert J; Hooke, Jeffrey A; Shriver, Craig D; Hu, Hai; Mitchell, Edith P; Freydin, Boris; Hyslop, Terry; Chervoneva, Inna; Rui, Hallgeir

    2016-01-01

    Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan–Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0

  17. Comparison of different immunochemical methods for the detection and quantification of hazelnut proteins in food products.

    PubMed

    Koppelman, S J; Knulst, A C; Koers, W J; Penninks, A H; Peppelman, H; Vlooswijk, R; Pigmans, I; van Duijn, G; Hessing, M

    1999-10-29

    Hazelnuts are widely used in the food industry owing to their nutritive value and taste. The amount of hazelnut present in a recipe is usually considered as a mark of quality. On the other hand, contamination of foods that normally do not contain hazelnuts is a threat for patients with a hazelnut allergy. For this reason, the availability of a method for the detection and quantification of hazelnuts in foods would be desirable. The objective of this study was to develop a method for the detection and quantification of minor amounts of hazelnut protein in food products that is potentially applicable for the food industry. Several immunochemical methods, e.g., immunoblotting and enzyme-linked immunosorbent assay (ELISA), were developed with antibodies from both hazelnut-sensitized patient sera and the sera of rabbits hyperimmunized with hazelnut protein. Immunoblotting appeared to be non-specific when the sera of patients were used as a source of antibodies. Using immunopurified antibodies from rabbits immunized with hazelnuts, immunoblotting became specific, but the sensitivity of this method was limited. Inhibition of IgE binding is a generally used test in clinical laboratories to establish contamination with hazelnuts. This approach is sensitive and specific, but not readily accessible for the food industry since patient serum is needed. Similar results in terms of sensitivity and specificity were obtained with a sandwich ELISA constructed with an immunopurified antibody from rabbits sensitized to hazelnuts. No substantial cross-reactivity with other nuts, legumes or other food constituents was observed, and concentrations as low as 5 ng/ml, corresponding to 1 ppm in food products, were detected. In a field test, several consumer products regarded to be free of hazelnuts were shown to contain traces of hazelnut. This sandwich ELISA constructed with immunopurified antibodies from rabbits sensitized with hazelnut protein is a sensitive and specific method to detect

  18. Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles.

    PubMed

    Walkiewicz, Marcin P; Morral, Nuria; Engel, Daniel A

    2009-08-01

    Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay.

  19. Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles.

    PubMed

    Walkiewicz, Marcin P; Morral, Nuria; Engel, Daniel A

    2009-08-01

    Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay. PMID:19406166

  20. Development of antigen capture ELISA for the quantification of EIAV p26 protein.

    PubMed

    Hu, Zhe; Chang, Hao; Ge, Man; Lin, Yuezhi; Wang, Xuefeng; Guo, Wei; Wang, Xiaojun

    2014-11-01

    An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

  1. Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

    PubMed Central

    Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

    2016-01-01

    The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

  2. Combining protein identification and quantification: C-terminal isotope-coded tagging using sulfanilic acid.

    PubMed

    Panchaud, Alexandre; Guillaume, Elisabeth; Affolter, Michael; Robert, Fabien; Moreillon, Philippe; Kussmann, Martin

    2006-01-01

    Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

  3. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    DOE PAGES

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test thismore » strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.« less

  4. Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

    SciTech Connect

    Melero, Cristina; Ollikainen, Noah; Harwood, Ian; Karpiak, Joel; Kortemme, Tanja

    2014-10-13

    Re-engineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. The context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

  5. FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues

    PubMed Central

    EL-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

    2016-01-01

    A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein

  6. FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

    PubMed

    El-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

    2016-01-01

    A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein

  7. Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins

    PubMed Central

    Tian, Bin; Harrison, Roland; Morton, James; Deb-Choudhury, Santanu

    2015-01-01

    Thaumatin-like proteins (TLPs) and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR) proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and determination of PR

  8. The identification of complete domains within protein sequences using accurate E-values for semi-global alignment

    PubMed Central

    Kann, Maricel G.; Sheetlin, Sergey L.; Park, Yonil; Bryant, Stephen H.; Spouge, John L.

    2007-01-01

    The sequencing of complete genomes has created a pressing need for automated annotation of gene function. Because domains are the basic units of protein function and evolution, a gene can be annotated from a domain database by aligning domains to the corresponding protein sequence. Ideally, complete domains are aligned to protein subsequences, in a ‘semi-global alignment’. Local alignment, which aligns pieces of domains to subsequences, is common in high-throughput annotation applications, however. It is a mature technique, with the heuristics and accurate E-values required for screening large databases and evaluating the screening results. Hidden Markov models (HMMs) provide an alternative theoretical framework for semi-global alignment, but their use is limited because they lack heuristic acceleration and accurate E-values. Our new tool, GLOBAL, overcomes some limitations of previous semi-global HMMs: it has accurate E-values and the possibility of the heuristic acceleration required for high-throughput applications. Moreover, according to a standard of truth based on protein structure, two semi-global HMM alignment tools (GLOBAL and HMMer) had comparable performance in identifying complete domains, but distinctly outperformed two tools based on local alignment. When searching for complete protein domains, therefore, GLOBAL avoids disadvantages commonly associated with HMMs, yet maintains their superior retrieval performance. PMID:17596268

  9. Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: implications for three-dimensional modeling.

    PubMed

    Bhaskara, Ramachandra M; Padhi, Amrita; Srinivasan, Narayanaswamy

    2014-07-01

    With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions.

  10. Toward more accurate ancestral protein genotype-phenotype reconstructions with the use of species tree-aware gene trees.

    PubMed

    Groussin, Mathieu; Hobbs, Joanne K; Szöllősi, Gergely J; Gribaldo, Simonetta; Arcus, Vickery L; Gouy, Manolo

    2015-01-01

    The resurrection of ancestral proteins provides direct insight into how natural selection has shaped proteins found in nature. By tracing substitutions along a gene phylogeny, ancestral proteins can be reconstructed in silico and subsequently synthesized in vitro. This elegant strategy reveals the complex mechanisms responsible for the evolution of protein functions and structures. However, to date, all protein resurrection studies have used simplistic approaches for ancestral sequence reconstruction (ASR), including the assumption that a single sequence alignment alone is sufficient to accurately reconstruct the history of the gene family. The impact of such shortcuts on conclusions about ancestral functions has not been investigated. Here, we show with simulations that utilizing information on species history using a model that accounts for the duplication, horizontal transfer, and loss (DTL) of genes statistically increases ASR accuracy. This underscores the importance of the tree topology in the inference of putative ancestors. We validate our in silico predictions using in vitro resurrection of the LeuB enzyme for the ancestor of the Firmicutes, a major and ancient bacterial phylum. With this particular protein, our experimental results demonstrate that information on the species phylogeny results in a biochemically more realistic and kinetically more stable ancestral protein. Additional resurrection experiments with different proteins are necessary to statistically quantify the impact of using species tree-aware gene trees on ancestral protein phenotypes. Nonetheless, our results suggest the need for incorporating both sequence and DTL information in future studies of protein resurrections to accurately define the genotype-phenotype space in which proteins diversify.

  11. Toward more accurate ancestral protein genotype-phenotype reconstructions with the use of species tree-aware gene trees.

    PubMed

    Groussin, Mathieu; Hobbs, Joanne K; Szöllősi, Gergely J; Gribaldo, Simonetta; Arcus, Vickery L; Gouy, Manolo

    2015-01-01

    The resurrection of ancestral proteins provides direct insight into how natural selection has shaped proteins found in nature. By tracing substitutions along a gene phylogeny, ancestral proteins can be reconstructed in silico and subsequently synthesized in vitro. This elegant strategy reveals the complex mechanisms responsible for the evolution of protein functions and structures. However, to date, all protein resurrection studies have used simplistic approaches for ancestral sequence reconstruction (ASR), including the assumption that a single sequence alignment alone is sufficient to accurately reconstruct the history of the gene family. The impact of such shortcuts on conclusions about ancestral functions has not been investigated. Here, we show with simulations that utilizing information on species history using a model that accounts for the duplication, horizontal transfer, and loss (DTL) of genes statistically increases ASR accuracy. This underscores the importance of the tree topology in the inference of putative ancestors. We validate our in silico predictions using in vitro resurrection of the LeuB enzyme for the ancestor of the Firmicutes, a major and ancient bacterial phylum. With this particular protein, our experimental results demonstrate that information on the species phylogeny results in a biochemically more realistic and kinetically more stable ancestral protein. Additional resurrection experiments with different proteins are necessary to statistically quantify the impact of using species tree-aware gene trees on ancestral protein phenotypes. Nonetheless, our results suggest the need for incorporating both sequence and DTL information in future studies of protein resurrections to accurately define the genotype-phenotype space in which proteins diversify. PMID:25371435

  12. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

    NASA Astrophysics Data System (ADS)

    Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

    2016-02-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally--a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process.

  13. Protein corona composition does not accurately predict hematocompatibility of colloidal gold nanoparticles.

    PubMed

    Dobrovolskaia, Marina A; Neun, Barry W; Man, Sonny; Ye, Xiaoying; Hansen, Matthew; Patri, Anil K; Crist, Rachael M; McNeil, Scott E

    2014-10-01

    Proteins bound to nanoparticle surfaces are known to affect particle clearance by influencing immune cell uptake and distribution to the organs of the mononuclear phagocytic system. The composition of the protein corona has been described for several types of nanomaterials, but the role of the corona in nanoparticle biocompatibility is not well established. In this study we investigate the role of nanoparticle surface properties (PEGylation) and incubation times on the protein coronas of colloidal gold nanoparticles. While neither incubation time nor PEG molecular weight affected the specific proteins in the protein corona, the total amount of protein binding was governed by the molecular weight of PEG coating. Furthermore, the composition of the protein corona did not correlate with nanoparticle hematocompatibility. Specialized hematological tests should be used to deduce nanoparticle hematotoxicity. From the clinical editor: It is overall unclear how the protein corona associated with colloidal gold nanoparticles may influence hematotoxicity. This study warns that PEGylation itself may be insufficient, because composition of the protein corona does not directly correlate with nanoparticle hematocompatibility. The authors suggest that specialized hematological tests must be used to deduce nanoparticle hematotoxicity.

  14. VORFFIP-driven dock: V-D2OCK, a fast and accurate protein docking strategy.

    PubMed

    Segura, Joan; Marín-López, Manuel Alejandro; Jones, Pamela F; Oliva, Baldo; Fernandez-Fuentes, Narcis

    2015-01-01

    The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers.

  15. Characterization and quantification of proteins secreted by single human embryos prior to implantation

    PubMed Central

    Poli, Maurizio; Ori, Alessandro; Child, Tim; Jaroudi, Souraya; Spath, Katharina; Beck, Martin; Wells, Dagan

    2015-01-01

    The use of in vitro fertilization (IVF) has revolutionized the treatment of infertility and is now responsible for 1–5% of all births in industrialized countries. During IVF, it is typical for patients to generate multiple embryos. However, only a small proportion of them possess the genetic and metabolic requirements needed in order to produce a healthy pregnancy. The identification of the embryo with the greatest developmental capacity represents a major challenge for fertility clinics. Current methods for the assessment of embryo competence are proven inefficient, and the inadvertent transfer of non-viable embryos is the principal reason why most IVF treatments (approximately two-thirds) end in failure. In this study, we investigate how the application of proteomic measurements could improve success rates in clinical embryology. We describe a procedure that allows the identification and quantification of proteins of embryonic origin, present in attomole concentrations in the blastocoel, the enclosed fluid-filled cavity that forms within 5-day-old human embryos. By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status. This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo. Our work paves the way for the development of “next-generation” embryo competence assessment strategies, based on functional proteomics. PMID:26471863

  16. Are current atomistic force fields accurate enough to study proteins in crowded environments?

    PubMed

    Petrov, Drazen; Zagrovic, Bojan

    2014-05-01

    The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the

  17. Are Current Atomistic Force Fields Accurate Enough to Study Proteins in Crowded Environments?

    PubMed Central

    Petrov, Drazen; Zagrovic, Bojan

    2014-01-01

    The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the

  18. Are current atomistic force fields accurate enough to study proteins in crowded environments?

    PubMed

    Petrov, Drazen; Zagrovic, Bojan

    2014-05-01

    The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD) simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded) and oxidatively damaged (unfolded) forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP) as well as indirectly shown for additional two (AMBER94, OPLS-AAL), and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields may distort the

  19. Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress

    PubMed Central

    Wu, Jianyong; Stevens, Jan F.; Maier, Claudia S.

    2012-01-01

    Acrolein exposure leads to the formation of protein-acrolein adducts. Protein modification by acrolein has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here we report an analytical strategy that enables the quantification of Michael-type protein adducts of acrolein in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein–acrolein adducts in relation to the unmodified protein thiol pool. Treatment of 3-month old rats with CCl4, an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct acrolein-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by acrolein were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by acrolein has potential value as an index of mitochondrial oxidative stress. PMID:21809440

  20. Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

    PubMed

    Niklasson, Markus; Ahlner, Alexandra; Andresen, Cecilia; Marsh, Joseph A; Lundström, Patrik

    2015-01-01

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

  1. Fast and Accurate Resonance Assignment of Small-to-Large Proteins by Combining Automated and Manual Approaches

    PubMed Central

    Niklasson, Markus; Ahlner, Alexandra; Andresen, Cecilia; Marsh, Joseph A.; Lundström, Patrik

    2015-01-01

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available. PMID:25569628

  2. Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

    PubMed

    Niklasson, Markus; Ahlner, Alexandra; Andresen, Cecilia; Marsh, Joseph A; Lundström, Patrik

    2015-01-01

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available. PMID:25569628

  3. Towards Accurate Residue-Residue Hydrophobic Contact Prediction for Alpha Helical Proteins Via Integer Linear Optimization

    PubMed Central

    Rajgaria, R.; McAllister, S. R.; Floudas, C. A.

    2008-01-01

    A new optimization-based method is presented to predict the hydrophobic residue contacts in α-helical proteins. The proposed approach uses a high resolution distance dependent force field to calculate the interaction energy between different residues of a protein. The formulation predicts the hydrophobic contacts by minimizing the sum of these contact energies. These residue contacts are highly useful in narrowing down the conformational space searched by protein structure prediction algorithms. The proposed algorithm also offers the algorithmic advantage of producing a rank ordered list of the best contact sets. This model was tested on four independent α-helical protein test sets and was found to perform very well. The average accuracy of the predictions (separated by at least six residues) obtained using the presented method was approximately 66% for single domain proteins. The average true positive and false positive distances were also calculated for each protein test set and they are 8.87 Å and 14.67 Å respectively. PMID:18767158

  4. Proteomic Quantification and Site-Mapping of S-Nitrosylated Proteins Using Isobaric iodoTMT Reagents

    PubMed Central

    2015-01-01

    S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease. PMID:24926564

  5. PROMALS3D web server for accurate multiple protein sequence and structure alignments.

    PubMed

    Pei, Jimin; Tang, Ming; Grishin, Nick V

    2008-07-01

    Multiple sequence alignments are essential in computational sequence and structural analysis, with applications in homology detection, structure modeling, function prediction and phylogenetic analysis. We report PROMALS3D web server for constructing alignments for multiple protein sequences and/or structures using information from available 3D structures, database homologs and predicted secondary structures. PROMALS3D shows higher alignment accuracy than a number of other advanced methods. Input of PROMALS3D web server can be FASTA format protein sequences, PDB format protein structures and/or user-defined alignment constraints. The output page provides alignments with several formats, including a colored alignment augmented with useful information about sequence grouping, predicted secondary structures and consensus sequences. Intermediate results of sequence and structural database searches are also available. The PROMALS3D web server is available at: http://prodata.swmed.edu/promals3d/. PMID:18503087

  6. A scalable and accurate method for classifying protein-ligand binding geometries using a MapReduce approach.

    PubMed

    Estrada, T; Zhang, B; Cicotti, P; Armen, R S; Taufer, M

    2012-07-01

    We present a scalable and accurate method for classifying protein-ligand binding geometries in molecular docking. Our method is a three-step process: the first step encodes the geometry of a three-dimensional (3D) ligand conformation into a single 3D point in the space; the second step builds an octree by assigning an octant identifier to every single point in the space under consideration; and the third step performs an octree-based clustering on the reduced conformation space and identifies the most dense octant. We adapt our method for MapReduce and implement it in Hadoop. The load-balancing, fault-tolerance, and scalability in MapReduce allow screening of very large conformation spaces not approachable with traditional clustering methods. We analyze results for docking trials for 23 protein-ligand complexes for HIV protease, 21 protein-ligand complexes for Trypsin, and 12 protein-ligand complexes for P38alpha kinase. We also analyze cross docking trials for 24 ligands, each docking into 24 protein conformations of the HIV protease, and receptor ensemble docking trials for 24 ligands, each docking in a pool of HIV protease receptors. Our method demonstrates significant improvement over energy-only scoring for the accurate identification of native ligand geometries in all these docking assessments. The advantages of our clustering approach make it attractive for complex applications in real-world drug design efforts. We demonstrate that our method is particularly useful for clustering docking results using a minimal ensemble of representative protein conformational states (receptor ensemble docking), which is now a common strategy to address protein flexibility in molecular docking. PMID:22658682

  7. Accurate retention time determination of co-eluting proteins in analytical chromatography by means of spectral data.

    PubMed

    Dismer, Florian; Hansen, Sigrid; Oelmeier, Stefan Alexander; Hubbuch, Jürgen

    2013-03-01

    Chromatography is the method of choice for the separation of proteins, at both analytical and preparative scale. Orthogonal purification strategies for industrial use can easily be implemented by combining different modes of adsorption. Nevertheless, with flexibility comes the freedom of choice and optimal conditions for consecutive steps need to be identified in a robust and reproducible fashion. One way to address this issue is the use of mathematical models that allow for an in silico process optimization. Although this has been shown to work, model parameter estimation for complex feedstocks becomes the bottleneck in process development. An integral part of parameter assessment is the accurate measurement of retention times in a series of isocratic or gradient elution experiments. As high-resolution analytics that can differentiate between proteins are often not readily available, pure protein is mandatory for parameter determination. In this work, we present an approach that has the potential to solve this problem. Based on the uniqueness of UV absorption spectra of proteins, we were able to accurately measure retention times in systems of up to four co-eluting compounds. The presented approach is calibration-free, meaning that prior knowledge of pure component absorption spectra is not required. Actually, pure protein spectra can be determined from co-eluting proteins as part of the methodology. The approach was tested for size-exclusion chromatograms of 38 mixtures of co-eluting proteins. Retention times were determined with an average error of 0.6 s (1.6% of average peak width), approximated and measured pure component spectra showed an average coefficient of correlation of 0.992.

  8. CRYSpred: accurate sequence-based protein crystallization propensity prediction using sequence-derived structural characteristics.

    PubMed

    Mizianty, Marcin J; Kurgan, Lukasz A

    2012-01-01

    Relatively low success rates of X-ray crystallography, which is the most popular method for solving proteins structures, motivate development of novel methods that support selection of tractable protein targets. This aspect is particularly important in the context of the current structural genomics efforts that allow for a certain degree of flexibility in the target selection. We propose CRYSpred, a novel in-silico crystallization propensity predictor that uses a set of 15 novel features which utilize a broad range of inputs including charge, hydrophobicity, and amino acid composition derived from the protein chain, and the solvent accessibility and disorder predicted from the protein sequence. Our method outperforms seven modern crystallization propensity predictors on three, independent from training dataset, benchmark test datasets. The strong predictive performance offered by the CRYSpred is attributed to the careful design of the features, utilization of the comprehensive set of inputs, and the usage of the Support Vector Machine classifier. The inputs utilized by CRYSpred are well-aligned with the existing rules-of-thumb that are used in the structural genomics studies. PMID:21919861

  9. CRYSpred: accurate sequence-based protein crystallization propensity prediction using sequence-derived structural characteristics.

    PubMed

    Mizianty, Marcin J; Kurgan, Lukasz A

    2012-01-01

    Relatively low success rates of X-ray crystallography, which is the most popular method for solving proteins structures, motivate development of novel methods that support selection of tractable protein targets. This aspect is particularly important in the context of the current structural genomics efforts that allow for a certain degree of flexibility in the target selection. We propose CRYSpred, a novel in-silico crystallization propensity predictor that uses a set of 15 novel features which utilize a broad range of inputs including charge, hydrophobicity, and amino acid composition derived from the protein chain, and the solvent accessibility and disorder predicted from the protein sequence. Our method outperforms seven modern crystallization propensity predictors on three, independent from training dataset, benchmark test datasets. The strong predictive performance offered by the CRYSpred is attributed to the careful design of the features, utilization of the comprehensive set of inputs, and the usage of the Support Vector Machine classifier. The inputs utilized by CRYSpred are well-aligned with the existing rules-of-thumb that are used in the structural genomics studies.

  10. Accurate design of megadalton-scale two-component icosahedral protein complexes.

    PubMed

    Bale, Jacob B; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-22

    Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines. PMID:27463675

  11. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    SciTech Connect

    Hong Xinguo; Hao Quan

    2009-01-15

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 deg. C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  12. Accurate Estimation of Protein Folding and Unfolding Times: Beyond Markov State Models.

    PubMed

    Suárez, Ernesto; Adelman, Joshua L; Zuckerman, Daniel M

    2016-08-01

    Because standard molecular dynamics (MD) simulations are unable to access time scales of interest in complex biomolecular systems, it is common to "stitch together" information from multiple shorter trajectories using approximate Markov state model (MSM) analysis. However, MSMs may require significant tuning and can yield biased results. Here, by analyzing some of the longest protein MD data sets available (>100 μs per protein), we show that estimators constructed based on exact non-Markovian (NM) principles can yield significantly improved mean first-passage times (MFPTs) for protein folding and unfolding. In some cases, MSM bias of more than an order of magnitude can be corrected when identical trajectory data are reanalyzed by non-Markovian approaches. The NM analysis includes "history" information, higher order time correlations compared to MSMs, that is available in every MD trajectory. The NM strategy is insensitive to fine details of the states used and works well when a fine time-discretization (i.e., small "lag time") is used. PMID:27340835

  13. Mathematical model accurately predicts protein release from an affinity-based delivery system.

    PubMed

    Vulic, Katarina; Pakulska, Malgosia M; Sonthalia, Rohit; Ramachandran, Arun; Shoichet, Molly S

    2015-01-10

    Affinity-based controlled release modulates the delivery of protein or small molecule therapeutics through transient dissociation/association. To understand which parameters can be used to tune release, we used a mathematical model based on simple binding kinetics. A comprehensive asymptotic analysis revealed three characteristic regimes for therapeutic release from affinity-based systems. These regimes can be controlled by diffusion or unbinding kinetics, and can exhibit release over either a single stage or two stages. This analysis fundamentally changes the way we think of controlling release from affinity-based systems and thereby explains some of the discrepancies in the literature on which parameters influence affinity-based release. The rate of protein release from affinity-based systems is determined by the balance of diffusion of the therapeutic agent through the hydrogel and the dissociation kinetics of the affinity pair. Equations for tuning protein release rate by altering the strength (KD) of the affinity interaction, the concentration of binding ligand in the system, the rate of dissociation (koff) of the complex, and the hydrogel size and geometry, are provided. We validated our model by collapsing the model simulations and the experimental data from a recently described affinity release system, to a single master curve. Importantly, this mathematical analysis can be applied to any single species affinity-based system to determine the parameters required for a desired release profile. PMID:25449806

  14. HAAD: A quick algorithm for accurate prediction of hydrogen atoms in protein structures.

    PubMed

    Li, Yunqi; Roy, Ambrish; Zhang, Yang

    2009-08-20

    Hydrogen constitutes nearly half of all atoms in proteins and their positions are essential for analyzing hydrogen-bonding interactions and refining atomic-level structures. However, most protein structures determined by experiments or computer prediction lack hydrogen coordinates. We present a new algorithm, HAAD, to predict the positions of hydrogen atoms based on the positions of heavy atoms. The algorithm is built on the basic rules of orbital hybridization followed by the optimization of steric repulsion and electrostatic interactions. We tested the algorithm using three independent data sets: ultra-high-resolution X-ray structures, structures determined by neutron diffraction, and NOE proton-proton distances. Compared with the widely used programs CHARMM and REDUCE, HAAD has a significantly higher accuracy, with the average RMSD of the predicted hydrogen atoms to the X-ray and neutron diffraction structures decreased by 26% and 11%, respectively. Furthermore, hydrogen atoms placed by HAAD have more matches with the NOE restraints and fewer clashes with heavy atoms. The average CPU cost by HAAD is 18 and 8 times lower than that of CHARMM and REDUCE, respectively. The significant advantage of HAAD in both the accuracy and the speed of the hydrogen additions should make HAAD a useful tool for the detailed study of protein structure and function. Both an executable and the source code of HAAD are freely available at http://zhang.bioinformatics.ku.edu/HAAD.

  15. Combining Evolutionary Information and an Iterative Sampling Strategy for Accurate Protein Structure Prediction.

    PubMed

    Braun, Tatjana; Koehler Leman, Julia; Lange, Oliver F

    2015-12-01

    Recent work has shown that the accuracy of ab initio structure prediction can be significantly improved by integrating evolutionary information in form of intra-protein residue-residue contacts. Following this seminal result, much effort is put into the improvement of contact predictions. However, there is also a substantial need to develop structure prediction protocols tailored to the type of restraints gained by contact predictions. Here, we present a structure prediction protocol that combines evolutionary information with the resolution-adapted structural recombination approach of Rosetta, called RASREC. Compared to the classic Rosetta ab initio protocol, RASREC achieves improved sampling, better convergence and higher robustness against incorrect distance restraints, making it the ideal sampling strategy for the stated problem. To demonstrate the accuracy of our protocol, we tested the approach on a diverse set of 28 globular proteins. Our method is able to converge for 26 out of the 28 targets and improves the average TM-score of the entire benchmark set from 0.55 to 0.72 when compared to the top ranked models obtained by the EVFold web server using identical contact predictions. Using a smaller benchmark, we furthermore show that the prediction accuracy of our method is only slightly reduced when the contact prediction accuracy is comparatively low. This observation is of special interest for protein sequences that only have a limited number of homologs.

  16. A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes.

    PubMed

    Wang, Michael Zhuo; Wu, Judy Qiju; Dennison, Jennifer B; Bridges, Arlene S; Hall, Stephen D; Kornbluth, Sally; Tidwell, Richard R; Smith, Philip C; Voyksner, Robert D; Paine, Mary F; Hall, James Edwin

    2008-10-01

    The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

  17. PETs: A Stable and Accurate Predictor of Protein-Protein Interacting Sites Based on Extremely-Randomized Trees.

    PubMed

    Xia, Bin; Zhang, Hong; Li, Qianmu; Li, Tao

    2015-12-01

    Protein-protein interaction (PPI) plays crucial roles in the performance of various biological processes. A variety of methods are dedicated to identify whether proteins have interaction residues, but it is often more crucial to recognize each amino acid. In practical applications, the stability of a prediction model is as important as its accuracy. However, random sampling, which is widely used in previous prediction models, often brings large difference between each training model. In this paper, a Predictor of protein-protein interaction sites based on Extremely-randomized Trees (PETs) is proposed to improve the prediction accuracy while maintaining the prediction stability. In PETs, a cluster-based sampling strategy is proposed to ensure the model stability: first, the training dataset is divided into subsets using specific features; second, the subsets are clustered using K-means; and finally the samples are selected from each cluster. Using the proposed sampling strategy, samples which have different types of significant features could be selected independently from different clusters. The evaluation shows that PETs is able to achieve better accuracy while maintaining a good stability. The source code and toolkit are available at https://github.com/BinXia/PETs.

  18. Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma.

    PubMed

    Zanaruddin, Sharifah Nurain Syed; Saleh, Amyza; Yang, Yi-Hsin; Hamid, Sharifah; Mustafa, Wan Mahadzir Wan; Khairul Bariah, A A N; Zain, Rosnah Binti; Lau, Shin Hin; Cheong, Sok Ching

    2013-03-01

    The presence of lymph node (LN) metastasis significantly affects the survival of patients with oral squamous cell carcinoma (OSCC). Successful detection and removal of positive LNs are crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the LNs, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis-positive LNs in the neck. We developed a molecular signature in the primary tumor that could predict LN metastasis in OSCC. A total of 211 cores from 55 individuals were included in the study. Eleven proteins were evaluated using immunohistochemical analysis in a tissue microarray. Of the 11 biomarkers evaluated using receiver operating curve analysis, epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2/neu), laminin, gamma 2 (LAMC2), and ras homolog family member C (RHOC) were found to be significantly associated with the presence of LN metastasis. Unsupervised hierarchical clustering-demonstrated expression patterns of these 4 proteins could be used to differentiate specimens that have positive LN metastasis from those that are negative for LN metastasis. Collectively, EGFR, HER-2/neu, LAMC2, and RHOC have a specificity of 87.5% and a sensitivity of 70%, with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated that the LN signature could independently predict disease-specific survival (P = .036). The 4-protein LN signature validated in an independent set of samples strongly suggests that it could reliably distinguish patients with LN metastasis from those who were metastasis-free and therefore could be a prognostic tool for the management of patients with OSCC.

  19. Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma.

    PubMed

    Zanaruddin, Sharifah Nurain Syed; Saleh, Amyza; Yang, Yi-Hsin; Hamid, Sharifah; Mustafa, Wan Mahadzir Wan; Khairul Bariah, A A N; Zain, Rosnah Binti; Lau, Shin Hin; Cheong, Sok Ching

    2013-03-01

    The presence of lymph node (LN) metastasis significantly affects the survival of patients with oral squamous cell carcinoma (OSCC). Successful detection and removal of positive LNs are crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the LNs, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis-positive LNs in the neck. We developed a molecular signature in the primary tumor that could predict LN metastasis in OSCC. A total of 211 cores from 55 individuals were included in the study. Eleven proteins were evaluated using immunohistochemical analysis in a tissue microarray. Of the 11 biomarkers evaluated using receiver operating curve analysis, epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2/neu), laminin, gamma 2 (LAMC2), and ras homolog family member C (RHOC) were found to be significantly associated with the presence of LN metastasis. Unsupervised hierarchical clustering-demonstrated expression patterns of these 4 proteins could be used to differentiate specimens that have positive LN metastasis from those that are negative for LN metastasis. Collectively, EGFR, HER-2/neu, LAMC2, and RHOC have a specificity of 87.5% and a sensitivity of 70%, with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated that the LN signature could independently predict disease-specific survival (P = .036). The 4-protein LN signature validated in an independent set of samples strongly suggests that it could reliably distinguish patients with LN metastasis from those who were metastasis-free and therefore could be a prognostic tool for the management of patients with OSCC. PMID:23026198

  20. Relative Quantification of Sites of Peptide and Protein Modification Using Size Exclusion Chromatography Coupled with Electron Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Xie, Boer; Sharp, Joshua S.

    2016-08-01

    One difficult problem in the analysis of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC separation using C18 reverse phase chromatography coupled with electron transfer dissociation (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixtures; however, the resolution of reverse phase chromatography greatly complicates quantification of isomeric modifications by ETD because of the chromatographic separation of peptides with identical modifications at different sequence positions. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences. This approach allows for the benefits of chromatographic separation of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using standard data-dependent acquisitions. Using this method, the relative amount of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a standard data-dependent acquisition experiment.

  1. The NIP7 protein is required for accurate pre-rRNA processing in human cells.

    PubMed

    Morello, Luis G; Hesling, Cédric; Coltri, Patrícia P; Castilho, Beatriz A; Rimokh, Ruth; Zanchin, Nilson I T

    2011-01-01

    Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman-Bodian-Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S-80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.

  2. Accurate quantification of total chromium and its speciation form Cr(VI) in water by ICP-DRC-IDMS and HPLC/ICP-DRC-IDMS.

    PubMed

    Markiewicz, Barbara; Komorowicz, Izabela; Barałkiewicz, Danuta

    2016-05-15

    Two analytical procedures have been developed for the determination of total chromium (TCr) and its highly toxic species, i.e. Cr(VI) in water samples using the following methods: inductively coupled plasma dynamic reaction cell isotope dilution mass spectrometry (ICP-DRC-IDMS) and high performance liquid chromatography inductively coupled plasma dynamic reaction cell isotope dilution mass spectrometry (HPLC/ICP-DRC-IDMS). Spectral interferences, predominantly occurring in chromium determination, were removed using a dynamic reaction cell (DRC). The presented procedures facilitate the quantification of trace amounts - below 1 µg L(-1) of TCr and individual Cr species - in various water matrices including drinking water and still bottled water with different mineral composition. Special attention has been paid to the adequate preparation of isotopically enriched (53)Cr(VI) standard solution in order to avoid artifacts in chromium speciation. Both procedures were fully validated as well as establishing the traceability and estimation of the uncertainty of measurement were carried out. Application of all of the above mentioned elements and of the isotope dilution technique, which provides the highest quality of metrological traceability, allowed to obtain reliable and high quality results of chromium determination in water samples. Additionally, the comparison of two methods: HPLC/ICP-DRC-MS and HPLC/ICP-DRC-IDMS for Cr(VI) determination, was submitted basing on the validation parameters. As a result, the lower values for these parameters were obtained using the second method. PMID:26992546

  3. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  4. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding

    PubMed Central

    Nissley, Daniel A.; Sharma, Ajeet K.; Ahmed, Nabeel; Friedrich, Ulrike A.; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P.

    2016-01-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally—a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process. PMID:26887592

  5. Accurate prediction of cellular co-translational folding indicates proteins can switch from post- to co-translational folding.

    PubMed

    Nissley, Daniel A; Sharma, Ajeet K; Ahmed, Nabeel; Friedrich, Ulrike A; Kramer, Günter; Bukau, Bernd; O'Brien, Edward P

    2016-01-01

    The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally--a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process. PMID:26887592

  6. Multi-reference-based multiple alignment statistics enables accurate protein-particle pickup from noisy images.

    PubMed

    Kawata, Masaaki; Sato, Chikara

    2013-04-01

    Data mining from noisy data/images is one of the most important themes in modern science and technology. Statistical image processing is a promising technique for analysing such data. Automation of particle pickup from noisy electron micrographs is essential, especially when improvement of the resolution of single particle analysis requires a huge number of particle images. For such a purpose, reference-based matching using primary three-dimensional (3D) model projections is mainly adopted. In the matching, however, the highest peaks of the correlation may not accurately indicate particles when the image is very noisy. In contrast, the density and the heights of the peaks should reflect the probability distribution of the particles. To statistically determine the particle positions from the peak distributions, we have developed a density-based peak search followed by a peak selection based on average peak height, using multi-reference alignment (MRA). Its extension, using multi-reference multiple alignment (MRMA), was found to enable particle pickup at higher accuracy even from extremely noisy images with a signal-to-noise ratio of 0.001. We refer to these new methods as stochastic pickup with MRA (MRA-StoPICK) or with MRMA (MRMA-StoPICK). MRMA-StoPICK has a higher pickup accuracy and furthermore, is almost independent of parameter settings. They were successfully applied to cryo-electron micrographs of Rice dwarf virus. Because current computational resources and parallel data processing environments allow somewhat CPU-intensive MRA-StoPICK and MRMA-StoPICK to be performed in a short period, these methods are expected to allow high-resolution analysis of the 3D structure of particles.

  7. Quantification of Borrelia burgdorferi Membrane Proteins in Human Serum: A New Concept for Detection of Bacterial Infection.

    PubMed

    Cheung, Crystal S F; Anderson, Kyle W; Benitez, Kenia Y Villatoro; Soloski, Mark J; Aucott, John N; Phinney, Karen W; Turko, Illarion V

    2015-11-17

    The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. The low abundance of bacterial proteins in human serum during infection imposes a challenge for early proteomic detection of Lyme disease. To address this challenge, we propose to detect membrane proteins released from bacteria due to disruption of their plasma membrane triggered by the innate immune system. These membrane proteins can be separated from the bulk of serum proteins by high-speed centrifugation causing substantial sample enrichment prior to targeted protein quantification using multiple reaction monitoring mass spectrometry. This new approach was first applied to detection of B. burgdorferi membrane proteins supplemented in human serum. Our results indicated that detection of B. burgdorferi membrane proteins, which are ≈10(7) lower in abundance than major serum proteins, is feasible. Therefore, quantitative analysis was also carried out for serum samples from three patients with acute Lyme disease. We were able to demonstrate the detection of ospA, the major B. burgdorferi lipoprotein at the level of 4.0 fmol of ospA/mg of serum protein. The results confirm the concept and suggest that the proposed approach can be expanded to detect other bacterial infections in humans, particularly where existing diagnostics are unreliable. PMID:26491962

  8. A tool for selective inline quantification of co-eluting proteins in chromatography using spectral analysis and partial least squares regression.

    PubMed

    Brestrich, Nina; Briskot, Till; Osberghaus, Anna; Hubbuch, Jürgen

    2014-07-01

    Selective quantification of co-eluting proteins in chromatography is usually performed by offline analytics. This is time-consuming and can lead to late detection of irregularities in chromatography processes. To overcome this analytical bottleneck, a methodology for selective protein quantification in multicomponent mixtures by means of spectral data and partial least squares regression was presented in two previous studies. In this paper, a powerful integration of software and chromatography hardware will be introduced that enables the applicability of this methodology for a selective inline quantification of co-eluting proteins in chromatography. A specific setup consisting of a conventional liquid chromatography system, a diode array detector, and a software interface to Matlab® was developed. The established tool for selective inline quantification was successfully applied for a peak deconvolution of a co-eluting ternary protein mixture consisting of lysozyme, ribonuclease A, and cytochrome c on SP Sepharose FF. Compared to common offline analytics based on collected fractions, no loss of information regarding the retention volumes and peak flanks was observed. A comparison between the mass balances of both analytical methods showed, that the inline quantification tool can be applied for a rapid determination of pool yields. Finally, the achieved inline peak deconvolution was successfully applied to make product purity-based real-time pooling decisions. This makes the established tool for selective inline quantification a valuable approach for inline monitoring and control of chromatographic purification steps and just in time reaction on process irregularities.

  9. Accurate quantification of sphingosine-1-phosphate in normal and Fabry disease plasma, cells and tissues by LC-MS/MS with (13)C-encoded natural S1P as internal standard.

    PubMed

    Mirzaian, Mina; Wisse, Patrick; Ferraz, Maria J; Marques, André R A; Gabriel, Tanit L; van Roomen, Cindy P A A; Ottenhoff, Roelof; van Eijk, Marco; Codée, Jeroen D C; van der Marel, Gijsbert A; Overkleeft, Herman S; Aerts, Johannes M

    2016-08-01

    We developed a mass spectrometric procedure to quantify sphingosine-1-phosphate (S1P) in biological materials. The use of newly synthesized (13)C5 C18-S1P and commercial C17-S1P as internal standards rendered very similar results with respect to linearity, limit of detection and limit of quantitation. Caution is warranted with determination of plasma S1P levels. Earlier it was reported that S1P is elevated in plasma of Fabry disease patients. We investigated this with the improved quantification. No clear conclusion could be drawn for patient plasma samples given the lack of uniformity of blood collection and plasma preparation. To still obtain insight, plasma and tissues were identically collected from α-galactosidase A deficient Fabry mice and matched control animals. No significant difference was observed in plasma S1P levels. A significant 2.3 fold increase was observed in kidney of Fabry mice, but not in liver and heart. Comparative analysis of S1P in cultured fibroblasts from normal subjects and classically affected Fabry disease males revealed no significant difference. In conclusion, accurate quantification of S1P in biological materials is feasible by mass spectrometry using the internal standards (13)C5 C18-S1P or C17-S1P. Significant local increases of S1P in the kidney might occur in Fabry disease as suggested by the mouse model. PMID:27221202

  10. Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes

    PubMed Central

    Victora, Andrea; Möller, Heiko M.; Exner, Thomas E.

    2014-01-01

    NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3–0.6 ppm and correlation coefficients (r2) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization. PMID:25404135

  11. Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve.

    PubMed

    Charette, Steve J; Lambert, Herman; Nadeau, Philippe J; Landry, Jacques

    2010-02-28

    Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.

  12. Characterization and quantification of grape variety by means of shikimic acid concentration and protein fingerprint in still white wines.

    PubMed

    Chabreyrie, David; Chauvet, Serge; Guyon, François; Salagoïty, Marie-Hélène; Antinelli, Jean-François; Medina, Bernard

    2008-08-27

    Protein profiles, obtained by high-performance capillary electrophoresis (HPCE) on white wines previously dialyzed, combined with shikimic acid concentration and multivariate analysis, were used for the determination of grape variety composition of a still white wine. Six varieties were studied through monovarietal wines elaborated in the laboratory: Chardonnay (24 samples), Chenin (24), Petit Manseng (7), Sauvignon (37), Semillon (24), and Ugni Blanc (9). Homemade mixtures were elaborated from authentic monovarietal wines according to a Plackett-Burman sampling plan. After protein peak area normalization, a matrix was elaborated containing protein results of wines (mixtures and monovarietal). Partial least-squares processing was applied to this matrix allowing the elaboration of a model that provided a varietal quantification precision of around 20% for most of the grape varieties studied. The model was applied to commercial samples from various geographical origins, providing encouraging results for control purposes.

  13. Complementary mass spectrometric techniques for the quantification of the protein corona: a case study on gold nanoparticles and human serum proteins.

    PubMed

    Fernández-Iglesias, Nerea; Bettmer, Jörg

    2015-09-14

    Once nanoparticles enter a biological system, it is known that their surface is instantly covered by the biomolecules present with preference to proteins. This protein corona has been a subject of numerous studies in order to reveal its composition. Besides that, growing interest exists in its quantitative determination in order to gain a deeper insight into the nature of these nanoparticle-protein bioconjugates. Only a few analytical methods are available nowadays, so the aim of this study is to provide a reliable and alternative methodology for the quantification of the protein corona. The suggested approach is based on the assumption that the total protein content within the corona can be correlated to its sulfur concentration due to the presence of cysteine and methionine as sulfur-containing amino acids. Once the most abundant proteins had been identified with the use of gel electrophoresis with subsequent peptide analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), the isolated nanoparticle-protein conjugates were subjected to total analysis of sulfur and the corresponding metal being present in the nanoparticles by inductively coupled plasma-mass spectrometry (ICP-MS). The concept is exemplarily demonstrated on citrate-stabilized gold nanoparticles (GNPs) incubated with human serum. Two different purification procedures were tested in order to isolate the sought bioconjugates. 26 most abundant proteins could be identified and an average of approximately 40 S atoms per protein was calculated and used for further studies. ICP-MS analyses of S/Au ratios served for the quantification of the protein corona revealing an absolute number of proteins bound to the incubated GNPs. Two main results could be obtained for this specific system under the chosen experimental conditions: the number of proteins per GNP decreased with their size from 10 nm to 60 nm and the obtained values suggested that the protein corona in this specific case was

  14. Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards

    PubMed Central

    Kramer, Gertjan; Woolerton, Yvonne; van Straalen, Jan P.; Vissers, Johannes P. C.; Dekker, Nick; Langridge, James I.; Beynon, Robert J.; Speijer, Dave; Sturk, Auguste; Aerts, Johannes M. F. G.

    2015-01-01

    Background Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. Results Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL—40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72–0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. Conclusions This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins. PMID:26474480

  15. Accurate mass determination, quantification and determination of detection limits in liquid chromatography-high-resolution time-of-flight mass spectrometry: challenges and practical solutions.

    PubMed

    Vergeynst, Leendert; Van Langenhove, Herman; Joos, Pieter; Demeestere, Kristof

    2013-07-30

    Uniform guidelines for the data processing and validation of qualitative and quantitative multi-residue analysis using full-spectrum high-resolution mass spectrometry are scarce. Through systematic research, optimal mass accuracy and sensitivity are obtained after refining the post-processing of the HRMS data. For qualitative analysis, transforming the raw profile spectra to centroid spectra is recommended resulting in a 2.3 fold improved precision on the accurate mass determination of spectrum peaks. However, processing centroid data for quantitative purposes could lead to signal interruption when too narrow mass windows are applied for the construction of extracted ion chromatograms. Therefore, peak integration on the raw profile data is recommended. An optimal width of the mass window of 50 ppm, which is a trade-off between sensitivity and selectivity, was obtained for a TOF instrument providing a resolving power of 20,000 at full width at half maximum (FWHM). For the validation of HRMS analytical methods, widespread concepts such as the signal-to-noise ratios for the determination of decision limits and detection capabilities have shown to be not always applicable because in some cases almost no noise can be detected anymore. A statistical methodology providing a reliable alternative is extended and applied. PMID:23856232

  16. Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.

    PubMed

    Han, Bingnan; Stevens, Jan F; Maier, Claudia S

    2007-05-01

    An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.

  17. Targeted absolute quantification of intact proteins by reversed phase liquid chromatography-mass spectrometry, charge reduced electrospray, and condensation particle counting.

    PubMed

    Adou, Kouame; Johnston, Murray V; Dykins, John L

    2012-08-21

    A novel approach involving the use of reversed phase liquid chromatography-mass spectrometry (RPLC-MS), charge reduced electrospray (CRES), and condensation particle counting (CPC) for the absolute quantification of intact proteins in liquid solutions is introduced. Under analysis conditions optimized for the quantification of select proteins within their predetermined linear ranges, a set of at least five protein standards with molecular weights (MW) spanning the dynamic ranges of both a quadrupole time-of-flight (QTOF) MS and a suitably selected RPLC column is used to generate a calibration curve of CPC detection efficiency (DE) as a function of the square root of MW. Next, the sample of interest is analyzed, and from the MS-generated MW data, the DE of each target protein is determined from the calibration curve. On the basis of MW, DE, and number concentration (molecules/unit volume), absolute quantification is achieved for each protein of interest. Application of this approach to the absolute quantification of cytochrome C (as target compound) in a commercial protein mixture is demonstrated with a deviation of 8%, a coefficient of variation (CV) of 5%, and a quantification limit of 432 fmol. For nontarget components of the mixture (ribonuclease A, holotransferrin, and apomyoglobin), the percent deviation from the stated concentrations and the CV varied from 0.20 to 23 and from 4.1 to 18, respectively. Performance of the method was further assessed by analyzing a laboratory quality control mixture comprising 0.33 μM of cytochrome C. The calculated value was 0.34 (CV: 5.1%). Universal in essence, the new technique holds strong promise for the absolute quantification of select proteins in liquid samples under conditions of good peak resolution and stable baseline.

  18. A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

    PubMed Central

    Forment, Josep V.; Jackson, Stephen P.

    2016-01-01

    Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

  19. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

    NASA Astrophysics Data System (ADS)

    Meyer, Jesse G.; D'Souza, Alexandria K.; Sorensen, Dylan J.; Rardin, Matthew J.; Wolfe, Alan J.; Gibson, Bradford W.; Schilling, Birgit

    2016-09-01

    Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

  20. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

    NASA Astrophysics Data System (ADS)

    Meyer, Jesse G.; D'Souza, Alexandria K.; Sorensen, Dylan J.; Rardin, Matthew J.; Wolfe, Alan J.; Gibson, Bradford W.; Schilling, Birgit

    2016-11-01

    Post-translational modification of lysine residues by NƐ-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.

  1. Complementary mass spectrometric techniques for the quantification of the protein corona: a case study on gold nanoparticles and human serum proteins

    NASA Astrophysics Data System (ADS)

    Fernández-Iglesias, Nerea; Bettmer, Jörg

    2015-08-01

    Once nanoparticles enter a biological system, it is known that their surface is instantly covered by the biomolecules present with preference to proteins. This protein corona has been a subject of numerous studies in order to reveal its composition. Besides that, growing interest exists in its quantitative determination in order to gain a deeper insight into the nature of these nanoparticle-protein bioconjugates. Only a few analytical methods are available nowadays, so the aim of this study is to provide a reliable and alternative methodology for the quantification of the protein corona. The suggested approach is based on the assumption that the total protein content within the corona can be correlated to its sulfur concentration due to the presence of cysteine and methionine as sulfur-containing amino acids. Once the most abundant proteins had been identified with the use of gel electrophoresis with subsequent peptide analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), the isolated nanoparticle-protein conjugates were subjected to total analysis of sulfur and the corresponding metal being present in the nanoparticles by inductively coupled plasma-mass spectrometry (ICP-MS). The concept is exemplarily demonstrated on citrate-stabilized gold nanoparticles (GNPs) incubated with human serum. Two different purification procedures were tested in order to isolate the sought bioconjugates. 26 most abundant proteins could be identified and an average of approximately 40 S atoms per protein was calculated and used for further studies. ICP-MS analyses of S/Au ratios served for the quantification of the protein corona revealing an absolute number of proteins bound to the incubated GNPs. Two main results could be obtained for this specific system under the chosen experimental conditions: the number of proteins per GNP decreased with their size from 10 nm to 60 nm and the obtained values suggested that the protein corona in this specific case was

  2. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    NASA Astrophysics Data System (ADS)

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-10-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1–3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites.

  3. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    PubMed Central

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-01-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1–3 mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites. PMID:27708384

  4. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    PubMed

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-01

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  5. PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

    PubMed

    Stokes, Matthew P; Farnsworth, Charles L; Moritz, Albrecht; Silva, Jeffrey C; Jia, Xiaoying; Lee, Kimberly A; Guo, Ailan; Polakiewicz, Roberto D; Comb, Michael J

    2012-05-01

    Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in

  6. SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

    NASA Astrophysics Data System (ADS)

    Gabant, Guillaume; Boyer, Alain; Cadene, Martine

    2016-08-01

    Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in: (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM.

  7. Quantification of a Non-conventional Protein Secretion: The Low-Molecular-Weight FGF-2 Example.

    PubMed

    Arcondéguy, Tania; Touriol, Christian; Lacazette, Eric

    2016-01-01

    Quantification of secreted factors is most often measured with enzyme-linked immunosorbent assay (ELISA), Western Blot, or more recently with antibody arrays. However, some of these, like low-molecular-weight fibroblast growth factor-2 (LMW FGF-2; the 18 kDa form), exemplify a set of secreted but almost non-diffusible molecular actors. It has been proposed that phosphorylated FGF-2 is secreted via a non-vesicular mechanism and that heparan sulfate proteoglycans function as extracellular reservoir but also as actors for its secretion. Heparan sulfate is a linear sulfated polysaccharide present on proteoglycans found in the extracellular matrix or anchored in the plasma membrane (syndecan). Moreover the LMW FGF-2 secretion appears to be activated upon FGF-1 treatment. In order to estimate quantification of such factor export across the plasma membrane, technical approaches are presented (evaluation of LMW FGF-2: (1) secretion, (2) extracellular matrix reservoir, and (3) secretion modulation by surrounding factors) and the importance of such procedures in the comprehension of the biology of these growth factors is underlined. PMID:27665555

  8. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

    PubMed Central

    Kettenbach, Arminja N; Rush, John; Gerber, Scott A

    2013-01-01

    In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for ‘absolute quantification’) method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase. PMID:21293459

  9. Antibody-free workflows for protein quantification by LC-MS/MS.

    PubMed

    Wilffert, Daniel; Bischoff, Rainer; van de Merbel, Nico C

    2015-01-01

    Antibody-free approaches for quantitative LC-MS/MS-based protein bioanalysis are reviewed and critically evaluated, and compared with the more widely used immunoaffinity-based approaches. Antibody-free workflows will be divided into four groups and discussed in the following order: direct analysis of signature peptides after proteolytic digestion; enrichment of target proteins and signature peptides by fractionated protein precipitation; enrichment of target proteins and signature peptides by reversed-phase and ion-exchange solid-phase extraction; and enrichment of target proteins and signature peptides by (antibody-free) affinity-solid-phase extraction. PMID:25871591

  10. Identification and quantification of host proteins in the vesicular fluid of porcine Taenia solium cysticerci.

    PubMed

    Navarrete-Perea, José; Moguel, Bárbara; Mendoza-Hernández, Guillermo; Fragoso, Gladis; Sciutto, Edda; Bobes, Raúl J; Laclette, Juan P

    2014-08-01

    The host-parasite relationship in cestode infections is complex. One feature of this bidirectional molecular communication is the uptake of host proteins by the parasite. Here we describe the presence of several host proteins in the vesicular fluid of Taenia solium cysticerci dissected from the central nervous system and the skeletal muscle of naturally infected pigs. Using two-dimensional electrophoresis we compared the protein patterns of vesicular fluids of cysticerci vs. the sera of cysticercotic pigs. We found that the vesicular fluids of both groups of cysts showed 17 protein spots matching with the pig's sera spots. After mass spectrometry sequencing of these spots, five host proteins were identified: hemoglobin, albumin, serpin A3-8, haptoglobin, rho GTPase-activating protein 36-like. Three of the 17 spots corresponded to host protein fragments: hemoglobin, albumin and serpin A3-8. IgG heavy and light chains were also identified by Western blot using a specific antibody. Quantitative estimations indicated that the host proteins represented 11-13% of the protein content in the vesicular fluids. We also calculated the relative abundance of these host proteins in the vesicular fluids; all were represented in similar relative abundances as in host sera. This suggests that uptake of host proteins by cysticerci proceeds through an unspecific mechanism such as non-specific fluid pinocytosis.

  11. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    PubMed

    Gallego, Marta; Mora, Leticia; Concepción Aristoy, M; Toldrá, Fidel

    2016-04-01

    The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham.

  12. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate.

  13. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  14. Quantification of protein-ligand interactions by mass spectrometry, titration, and H/D exchange: PLIMSTEX.

    PubMed

    Zhu, Mei M; Rempel, Don L; Du, Zhaohui; Gross, Michael L

    2003-05-01

    Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.

  15. Identification and quantification of calcium-binding proteins in squid axoplasm

    SciTech Connect

    Krinks, M.H.; Klee, C.B.; Pant, H.C.; Gainer, H.

    1988-06-01

    The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. /sup 45/Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3) neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium-binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca/sup 2 +//kg axoplasm. /sup 125/I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca/sup 2 +//CaM protein kinase II subunits.

  16. Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems.

    PubMed

    Sauvant, D; Nozière, P

    2016-05-01

    The evolution of feeding systems for ruminants towards evaluation of diets in terms of multiple responses requires the updating of the calculation of nutrient supply to the animals to make it more accurate on aggregated units (feed unit, or UF, for energy and protein digestible in the intestine, or PDI, for metabolizable protein) and to allow prediction of absorbed nutrients. The present update of the French system is based on the building and interpretation through meta-analysis of large databases on digestion and nutrition of ruminants. Equations involved in the calculation of UF and PDI have been updated, allowing: (1) prediction of the out flow rate of particles and liquid depending on the level of intake and the proportion of concentrate, and the use of this in the calculation of ruminal digestion of protein and starch from in situ data; (2) the system to take into account the effects of the main factors of digestive interactions (level of intake, proportion of concentrate, rumen protein balance) on organic matter digestibility, energy losses in methane and in urine; (3) more accurate calculation of the energy available in the rumen and the efficiency of its use for the microbial protein synthesis. In this renewed model UF and PDI values of feedstuffs vary depending on diet composition, and intake level. Consequently, standard feed table values can be considered as being only indicative. It is thus possible to predict the nutrient supply on a wider range of diets more accurately and in particular to better integrate energy×protein interactions occurring in the gut.

  17. Quantification of the main digestive processes in ruminants: the equations involved in the renewed energy and protein feed evaluation systems.

    PubMed

    Sauvant, D; Nozière, P

    2016-05-01

    The evolution of feeding systems for ruminants towards evaluation of diets in terms of multiple responses requires the updating of the calculation of nutrient supply to the animals to make it more accurate on aggregated units (feed unit, or UF, for energy and protein digestible in the intestine, or PDI, for metabolizable protein) and to allow prediction of absorbed nutrients. The present update of the French system is based on the building and interpretation through meta-analysis of large databases on digestion and nutrition of ruminants. Equations involved in the calculation of UF and PDI have been updated, allowing: (1) prediction of the out flow rate of particles and liquid depending on the level of intake and the proportion of concentrate, and the use of this in the calculation of ruminal digestion of protein and starch from in situ data; (2) the system to take into account the effects of the main factors of digestive interactions (level of intake, proportion of concentrate, rumen protein balance) on organic matter digestibility, energy losses in methane and in urine; (3) more accurate calculation of the energy available in the rumen and the efficiency of its use for the microbial protein synthesis. In this renewed model UF and PDI values of feedstuffs vary depending on diet composition, and intake level. Consequently, standard feed table values can be considered as being only indicative. It is thus possible to predict the nutrient supply on a wider range of diets more accurately and in particular to better integrate energy×protein interactions occurring in the gut. PMID:26696120

  18. A single procedure for the accurate and precise quantification of the rare earth elements, Sc, Y, Th and Pb in dust and peat for provenance tracing in climate and environmental studies.

    PubMed

    Ferrat, Marion; Weiss, Dominik J; Strekopytov, Stanislav

    2012-05-15

    The geochemical provenancing of atmospheric dust deposited in terrestrial archives such as peat bogs using trace elements is central to the study of atmospheric deposition over the continents and at the heart of many climate and environmental studies. The use of a single digestion method on all sample types involved in such a study (dust archive and sources) minimizes the contribution of the total analytical error when comparing sample compositions and attributing a source to the deposited dust. To date, this factor is limiting progress in geographical areas where the compositional variations between the sources and within the archive are small. Here, seven microwave and hot plate digestion methods were tested on rock, soil and plant reference materials to establish a unique method optimizing precision and accuracy in all sample types. The best results were obtained with a hot plate closed-vessel digestion with 2 ml HF and 0.5 ml HNO(3) for 0.1g of sample, which allowed the precise, accurate and low blank quantification of the trace elements La-Yb, Sc, Y, Th and Pb by ICP-MS. This method was tested in a climate study in central Asia and temporal changes in the dominant dust source were for the first time successfully linked to changes in atmospheric circulation patterns above this region.

  19. Accurate LC-ESI-MS/MS quantification of 2'-deoxymugineic acid in soil and root related samples employing porous graphitic carbon as stationary phase and a ¹³C₄-labeled internal standard.

    PubMed

    Schindlegger, Yvonne; Oburger, Eva; Gruber, Barbara; Schenkeveld, Walter D C; Kraemer, Stephan M; Puschenreiter, Markus; Koellensperger, Gunda; Hann, Stephan

    2014-05-01

    For the first time the phytosiderophore 2'-deoxymugineic acid (DMA) could be accurately quantified by LC-MS/MS in plant and soil related samples. For this purpose a novel chromatographic method employing porous graphitic carbon as stationary phase combined with ESI-MS/MS detection in selected reaction monitoring was developed. Isotope dilution was implemented by using in-house synthesized DMA as external calibrant and ¹³C₄-labeled DMA as internal standard (concentration levels of standards 0.1-80 μM, determination coefficient of linear regression R² > 0.9995). Sample preparation involved acidification of the samples in order to obtain complete dissociation of metal-DMA complexes. Excellent matrix related LOD and LOQ depending on different experimental setups were obtained in the range of 3-34 nM and 11-113 nM, respectively. Standard addition experiments and the implementation of the internal ¹³C₄-DMA standard proved the accuracy of the quantification strategy even in complex matrices such as soil solution. The repeatability of the method, including sample preparation, expressed as short- and long term precision was below 4 and 5% RSD, respectively. Finally, application in the context of plant and soil research to samples from rhizosphere sampling via micro suction cups, from soil solutions and soil adsorption/extraction studies revealed a DMA concentration range from 0.1 to 235 μM.

  20. Quantification of β-region IgA monoclonal proteins - should we include immunochemical Hevylite® measurements? Point.

    PubMed

    Evans, Josie A R; Jenner, Ellen L; Carr Smith, Hugh D; Berlanga, Oscar; Harding, Stephen J

    2016-06-01

    Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.

  1. Stress-Inducible Protein 1 (STI1): Extracellular Vesicle Analysis and Quantification.

    PubMed

    Dias, Marcos Vinicios Salles; Martins, Vilma Regina; Hajj, Glaucia Noeli Maroso

    2016-01-01

    This chapter is derived from our experience in the study of stress-Inducible Protein 1 (STI1) in extracellular vesicles. We used different techniques to isolate, explore, and characterize the extracellular vesicles that contained this protein. Ultracentrifugation and gel chromatography were used to isolate extracellular vesicles of different sizes, nanotracking particle analysis (NTA) determined number and size of vesicles, while flow cytometry and ELISA were used to determine the specific protein content of vesicles. PMID:27665558

  2. Novel isotopic N, N-dimethyl leucine (iDiLeu) reagents enable absolute quantification of peptides and proteins using a standard curve approach

    PubMed Central

    Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

    2014-01-01

    Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive due to the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using Mass Differential Tags for Relative and Absolute Quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N,N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective due to their synthetic simplicity, and have increased throughput compared to previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error) while the second enables standard curve creation and analyte quantification in one run (<8% error). PMID:25377360

  3. Characterization of Nanoparticle Tracking Analysis for Quantification and Sizing of Submicron Particles of Therapeutic Proteins.

    PubMed

    Zhou, Chen; Krueger, Aaron B; Barnard, James G; Qi, Wei; Carpenter, John F

    2015-08-01

    Submicron particles may play important roles in therapeutic protein product quality, stability, and adverse effects in patients. However, quantitation of these particles has been challenging. Nanoparticle tracking analysis (NTA) is capable of both sizing and counting submicron particles. We investigated the effects of product and instrument parameters on NTA results for nanoparticle standards and therapeutic protein samples. To obtain proper particle size distributions, complete tracking numbers of at least 200 and 400 were required for latex nanobeads and protein nanoparticles, respectively. In addition, when set at suboptimal values, the minimum expected particle size parameter led to inaccurate sizing and counting for all particles types investigated. A syringe pump allowed for higher sampling volumes, and results were reproducible for nanoparticle sizing and counts at flow rates ≤7 μL/min. Finally, because therapeutic protein products are being formulated at relatively high protein concentrations, we investigated the effects of protein concentration on nanoparticle characterization. With high protein concentrations, nanoparticle sizing was not affected, whereas particle concentrations were significantly reduced. Linear relationships between particle count and dilution factor were obtained when a high protein concentration formulation was diluted into particle-free solutions at the same protein concentrations, but not when dilutions were made into buffer.

  4. Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis

    NASA Astrophysics Data System (ADS)

    Abdisalaam, Salim; Poudel, Milan; Chen, David J.; Alexandrakis, George

    2011-03-01

    The kinetics of most proteins involved in DNA damage sensing, signaling and repair following ionizing radiation exposure cannot be quantified by current live cell fluorescence microscopy methods. This is because most of these proteins, with only few notable exceptions, do not attach in large numbers at DNA damage sites to form easily detectable foci in microscopy images. As a result a high fluorescence background from freely moving and immobile fluorescent proteins in the nucleus masks the aggregation of proteins at sparse DNA damage sites. Currently, the kinetics of these repair proteins are studied by laser-induced damage and Fluorescence Recovery After Photobleaching that rely on the detectability of high fluorescence intensity spots of clustered DNA damage. We report on the use of Number and Brightness (N&B) analysis methods as a means to monitor kinetics of DNA repair proteins during sparse DNA damage created by γ-irradiation, which is more relevant to cancer treatment than laser-induced clustered damage. We use two key double strand break repair proteins, namely Ku 70/80 and the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), as specific examples to showcase the feasibility of the proposed methods to quantify dose-dependent kinetics for DNA repair proteins after exposure to γ-rays.

  5. Quantification and Classification of E. coli Proteome Utilization and Unused Protein Costs across Environments

    PubMed Central

    O’Brien, Edward J.; Utrilla, Jose; Palsson, Bernhard O.

    2016-01-01

    The costs and benefits of protein expression are balanced through evolution. Expression of un-utilized protein (that have no benefits in the current environment) incurs a quantifiable fitness costs on cellular growth rates; however, the magnitude and variability of un-utilized protein expression in natural settings is unknown, largely due to the challenge in determining environment-specific proteome utilization. We address this challenge using absolute and global proteomics data combined with a recently developed genome-scale model of Escherichia coli that computes the environment-specific cost and utility of the proteome on a per gene basis. We show that nearly half of the proteome mass is unused in certain environments and accounting for the cost of this unused protein expression explains >95% of the variance in growth rates of Escherichia coli across 16 distinct environments. Furthermore, reduction in unused protein expression is shown to be a common mechanism to increase cellular growth rates in adaptive evolution experiments. Classification of the unused protein reveals that the unused protein encodes several nutrient- and stress- preparedness functions, which may convey fitness benefits in varying environments. Thus, unused protein expression is the source of large and pervasive fitness costs that may provide the benefit of hedging against environmental change. PMID:27351952

  6. Quantification and Classification of E. coli Proteome Utilization and Unused Protein Costs across Environments.

    PubMed

    O'Brien, Edward J; Utrilla, Jose; Palsson, Bernhard O

    2016-06-01

    The costs and benefits of protein expression are balanced through evolution. Expression of un-utilized protein (that have no benefits in the current environment) incurs a quantifiable fitness costs on cellular growth rates; however, the magnitude and variability of un-utilized protein expression in natural settings is unknown, largely due to the challenge in determining environment-specific proteome utilization. We address this challenge using absolute and global proteomics data combined with a recently developed genome-scale model of Escherichia coli that computes the environment-specific cost and utility of the proteome on a per gene basis. We show that nearly half of the proteome mass is unused in certain environments and accounting for the cost of this unused protein expression explains >95% of the variance in growth rates of Escherichia coli across 16 distinct environments. Furthermore, reduction in unused protein expression is shown to be a common mechanism to increase cellular growth rates in adaptive evolution experiments. Classification of the unused protein reveals that the unused protein encodes several nutrient- and stress- preparedness functions, which may convey fitness benefits in varying environments. Thus, unused protein expression is the source of large and pervasive fitness costs that may provide the benefit of hedging against environmental change. PMID:27351952

  7. Detergent quantification in membrane protein samples and its application to crystallization experiments.

    PubMed

    Prince, Chelsy C; Jia, Zongchao

    2013-12-01

    The structural characterization of membrane proteins remains a challenging field, largely because the use of stabilizing detergents is required. Researchers must first select a suitable detergent for the solubility and stability of their protein during in vitro studies. In addition, an appropriate concentration of detergent in membrane protein samples can be essential for protein solubility, stability, and experimental success. For example, in membrane protein crystallography, detergent concentration in the crystallization drop can be a critical parameter influencing crystal growth. Over the past decade, multiple techniques have been developed for the measurement of detergent concentration using a wide variety of strategies. These methods include colorimetric reactions, which target specific detergent classes, and analytical techniques applicable to a wide variety of detergents. This review will summarize and discuss the available options. It will be a useful resource to those selecting a strategy that best fits their experimental requirements and available instruments.

  8. A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

    SciTech Connect

    Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.; Metz, Thomas O.; Pounds, Joel G.; Rodland, Karin D.; Shukla, Anil K.; Smith, Richard D.; Waters, Katrina M.; McDermott, Jason E.; Webb-Robertson, Bobbie-Jo M.

    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 from 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.

  9. Antibody-independent Targeted Quantification of TMPRSS2-ERG Fusion Protein Products in Prostate Cancer

    SciTech Connect

    He, Jintang; Sun, Xuefei; Shi, Tujin; Schepmoes, Athena A.; Fillmore, Thomas L.; Petyuk, Vladislav A.; Xie, Fang; Zhao, Rui; Gritsenko, Marina A.; Yang, Feng; Kitabayashi, Naoki; Chae, Sung Suk; Rubin, Mark; Siddiqui, Javed; Wei, John; Chinnaiyan, Arul M.; Qian, Weijun; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Liu, Tao; Camp, David G.

    2014-10-01

    Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. The studies on TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies or an antibody-independent method that is sufficiently sensitive for detecting the truncated ERG protein products resulting from TMPRSS2-ERG gene fusions and alternative splicing. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays led to confident detection of 6 unique ERG peptides in either the TMPRSS2-ERG positive cell lines or tissues but not in the negative controls, indicating that ERG protein expression is highly correlated with TMPRSS2-ERG gene rearrangements. Significantly, our results demonstrated for the first time that at least two groups of ERG protein isoforms were simultaneously expressed at variable levels in TMPRSS2-ERG positive samples as evidenced by concomitant detection of two mutually exclusive peptides. Three peptides shared across almost all fusion protein products were determined to be the most abundant peptides, and hence can be used as “signature” peptides for detecting ERG overexpression resulting from TMPRSS2-ERG gene fusion. These PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products, thus improving our understanding of the role of TMPRSS2-ERG gene fusion in the biology of prostate cancer.

  10. Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity.

    PubMed Central

    Roberts, D. W.; Bucci, T. J.; Benson, R. W.; Warbritton, A. R.; McRae, T. A.; Pumford, N. R.; Hinson, J. A.

    1991-01-01

    Acetaminophen overdose causes severe hepatotoxicity in humans and laboratory animals, presumably by metabolism to N-acetyl-p-benzoquinone imine: and binding to cysteine groups as 3-(cystein-S-yl)acetaminophen-protein adduct. Antiserum specific for the adduct was used immunohistochemically to demonstrate the formation, distribution, and concentration of this specific adduct in livers of treated mice and was correlated with cell injury as a function of dose and time. Within the liver lobule, immunohistochemically demonstrable adduct occurred in a temporally progressive, central-to-peripheral pattern. There was concordance between immunohistochemical staining and quantification of the adduct in hepatic 10,000g supernate, using a quantitative particle concentration fluorescence immunoassay. Findings include: 1) immunochemically detectable adduct before the appearance of centrilobular necrosis, 2) distinctive lobular zones of adduct localization with subsequent depletion during the progression of toxicity, 3) drug-protein binding in hepatocytes at subhepatotoxic doses and before depletion of total hepatic glutathione, 4) immunohistochemical evidence of drug binding in the nucleus, and 5) adduct in metabolically active and dividing hepatocytes and in macrophagelike cells in the regenerating liver. Images Figure 2 Figure 4 PMID:1992763

  11. Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.

    PubMed

    Karbowski, Mariusz; Cleland, Megan M; Roelofs, Brian A

    2014-01-01

    Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis, and stress-induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assays rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

  12. A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

    PubMed Central

    Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

    2013-01-01

    A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

  13. Dual transcript and protein quantification in a massive single cell array.

    PubMed

    Park, Seung-Min; Lee, Jae Young; Hong, Soongweon; Lee, Sang Hun; Dimov, Ivan K; Lee, Hojae; Suh, Susie; Pan, Qiong; Li, Keyu; Wu, Anna M; Mumenthaler, Shannon M; Mallick, Parag; Lee, Luke P

    2016-10-01

    Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms. PMID:27546183

  14. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

    PubMed

    Etheridge, Thomas J; Boulineau, Rémi L; Herbert, Alex; Watson, Adam T; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A; Klenerman, David; Lee, Steven F; Carr, Antony M

    2014-10-29

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

  15. Quantification of protein interactions and solution transport using high-density GMR sensor arrays.

    PubMed

    Gaster, Richard S; Xu, Liang; Han, Shu-Jen; Wilson, Robert J; Hall, Drew A; Osterfeld, Sebastian J; Yu, Heng; Wang, Shan X

    2011-05-01

    Monitoring the kinetics of protein interactions on a high-density sensor array is vital to drug development and proteomic analysis. Label-free kinetic assays based on surface plasmon resonance are the current gold standard, but they have poor detection limits, suffer from non-specific binding, and are not amenable to high-throughput analyses. Here, we show that magnetically responsive nanosensors that have been scaled to over 100,000 sensors per cm² can be used to measure the binding kinetics of various proteins with high spatial and temporal resolution. We present an analytical model that describes the binding of magnetically labelled antibodies to proteins that are immobilized on the sensor surface. This model is able to quantify the kinetics of antibody-antigen binding at sensitivities as low as 20 zeptomoles of solute.

  16. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

    PubMed Central

    Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

    2014-01-01

    Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

  17. SynPAnal: software for rapid quantification of the density and intensity of protein puncta from fluorescence microscopy images of neurons.

    PubMed

    Danielson, Eric; Lee, Sang H

    2014-01-01

    Continuous modification of the protein composition at synapses is a driving force for the plastic changes of synaptic strength, and provides the fundamental molecular mechanism of synaptic plasticity and information storage in the brain. Studying synaptic protein turnover is not only important for understanding learning and memory, but also has direct implication for understanding pathological conditions like aging, neurodegenerative diseases, and psychiatric disorders. Proteins involved in synaptic transmission and synaptic plasticity are typically concentrated at synapses of neurons and thus appear as puncta (clusters) in immunofluorescence microscopy images. Quantitative measurement of the changes in puncta density, intensity, and sizes of specific proteins provide valuable information on their function in synaptic transmission, circuit development, synaptic plasticity, and synaptopathy. Unfortunately, puncta quantification is very labor intensive and time consuming. In this article, we describe a software tool designed for the rapid semi-automatic detection and quantification of synaptic protein puncta from 2D immunofluorescence images generated by confocal laser scanning microscopy. The software, dubbed as SynPAnal (for Synaptic Puncta Analysis), streamlines data quantification for puncta density and average intensity, thereby increases data analysis throughput compared to a manual method. SynPAnal is stand-alone software written using the JAVA programming language, and thus is portable and platform-free.

  18. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    PubMed

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. PMID:23894077

  19. Quantification of proteins in urine samples using targeted mass spectrometry methods.

    PubMed

    Khristenko, Nina; Domon, Bruno

    2015-01-01

    Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements.

  20. Quantification of proteins in urine samples using targeted mass spectrometry methods.

    PubMed

    Khristenko, Nina; Domon, Bruno

    2015-01-01

    Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements. PMID:25384748

  1. Constrained Selected Reaction Monitoring: Quantification of selected post-translational modifications and protein isoforms

    PubMed Central

    Liu, Xiaoqian; Jin, Zhicheng; O’Brien, Richard; Bathon, Joan; Dietz, Harry C.; Grote, Eric; Van Eyk, Jennifer E.

    2014-01-01

    Selected reaction monitoring (SRM) is a mass spectrometry method that can target signature peptides to provide for the detection and quantitation of specific proteins in complex biological samples. When quantifying a protein, peptides are generated using a specific protease such as trypsin, allowing the choice of signature peptides with robust signals. In contrast, signature peptide selection can be constrained when the goal is to monitor a specific post-translational modification (PTM) or protein isoform as the signature peptide must include the amino acid residue(s) of PTM attachment or sequence variation. This can force the selection of a signature peptide with a weak SRM response or one that is confounded by high background. In this article, additional steps that can be optimized to maximize peptide selection and assay performance of constrained SRM assays are discussed including tuning instrument parameters, fragmenting product ions, using a different protease, and enriching the sample. Examples are provided for selection of phosphorylated or citrullinated peptides and protein isoforms. PMID:23523700

  2. Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics.

    PubMed

    Kleckner, Ian R; Gollnick, Paul; Foster, Mark P

    2012-01-13

    The trp RNA-binding attenuation protein (TRAP) is a paradigmatic allosteric protein that regulates the tryptophan biosynthetic genes associated with the trp operon in bacilli. The ring-shaped 11-mer TRAP is activated for recognition of a specific trp-mRNA target by binding up to 11 tryptophan molecules. To characterize the mechanisms of tryptophan-induced TRAP activation, we have performed methyl relaxation dispersion (MRD) nuclear magnetic resonance (NMR) experiments that probe the time-dependent structure of TRAP in the microsecond-to-millisecond "chemical exchange" time window. We find significant side chain flexibility localized to the RNA and tryptophan binding sites of the apo protein and that these dynamics are dramatically reduced upon ligand binding. Analysis of the MRD NMR data provides insights into the structural nature of transiently populated conformations sampled in solution by apo TRAP. The MRD data are inconsistent with global two-state exchange, indicating that conformational sampling in apo TRAP is asynchronous. These findings imply a temporally heterogeneous population of structures that are incompatible with RNA binding and substantiate the study of TRAP as a paradigm for probing and understanding essential dynamics in allosteric, regulatory proteins. PMID:22115774

  3. Elemental Analysis and Protein Quantification of Raw Natural Rubber of IAC Series 400 Clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein, nitrogen and sulfur contents were investigated for natural rubber from commercial Hevea, synthetic polyisoprene, and new IAC clones from Mococa city (IAC 405, 406, 410, 413, and 420), Jaú city (IAC 400, 401, 402, and 417), and from RRIM 600 clone (used as a control in both cases). IAC 405 a...

  4. Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

    SciTech Connect

    He, Jintang; Schepmoes, Athena A.; Shi, Tujin; Wu, Chaochao; Fillmore, Thomas L.; Gao, Yuqian; Smith, Richard D.; Qian, Weijun; Rodland, Karin D.; Liu, Tao; Camp, David G.; Rastogi, Anshu; Tan, Shyh-Han; Yan, Wusheng; Mohamed, Ahmed A.; Huang, Wei; Banerjee, Sreedatta; Kagan, Jacob; Srivastava, Sudhir; McLeod, David; Srivastava, Shiv; Petrovics, Gyorgy; Dobi, Albert; Srinivasan, Alagarsamy

    2015-01-01

    Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: Therefore, an antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was around approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house enzyme-linked immunosorbent assay (ELISA) was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostics and prognostics assays for prostate cancer given their sensitivity, specificity, and reproducibility.

  5. Analytical platform evaluation for quantification of ERG in prostate cancer using protein and mRNA detection methods

    DOE PAGES

    He, Jintang; Schepmoes, Athena A.; Shi, Tujin; Wu, Chaochao; Fillmore, Thomas L.; Gao, Yuqian; Smith, Richard D.; Qian, Weijun; Rodland, Karin D.; Liu, Tao; et al

    2015-01-01

    Background: The established methods for detecting prostate cancer (CaP) are based on tests using PSA (blood), PCA3 (urine), and AMACR (tissue) as biomarkers in patient samples. The demonstration of ERG oncoprotein overexpression due to gene fusion in CaP has thus provided ERG as an additional biomarker. Based on this, we hypothesized that ERG protein quantification methods can be of use in the diagnosis of prostate cancer. Methods: Therefore, an antibody-free assay for ERG3 protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERGmore » negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment). Results: Recombinant ERG3 protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was around approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine. The sensitivity of the in-house enzyme-linked immunosorbent assay (ELISA) was similar to the PRISM-SRM assay, with detection of 30 pg of purified recombinant ERG3 protein and 10,000 VCaP cells. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 100 VCaP cells, in comparison to NanoString methodologies which detected ERG from 10,000 cells. Conclusions: Based on this data, we propose that the detection of both ERG transcriptional products with RNA-based assays, as well as protein products of ERG using PRISM-SRM assays, may be of clinical value in developing diagnostics and prognostics assays for prostate cancer given their sensitivity, specificity, and reproducibility.« less

  6. Quantification of the affinities and kinetics of protein interactions using silicon nanowire biosensors

    PubMed Central

    Duan, Xuexin; Li, Yue; Rajan, Nitin K.; Routenberg, David A.; Modis, Yorgo; Reed, Mark A.

    2014-01-01

    Monitoring the binding affinities and kinetics of protein interactions is important in clinical diagnostics and drug development because such information is used to identify new therapeutic candidates. Surface plasmon resonance is at present the standard method used for such analysis, but this is limited by low sensitivity and low-throughput analysis. Here, we show that silicon nanowire field-effect transistors can be used as biosensors to measure protein–ligand binding affinities and kinetics with sensitivities down to femtomolar concentrations. Based on this sensing mechanism, we develop an analytical model to calibrate the sensor response and quantify the molecular binding affinities of two representative protein–ligand binding pairs. The rate constant of the association and dissociation of the protein–ligand pair is determined by monitoring the reaction kinetics, demonstrating that silicon nanowire field-effect transistors can be readily used as high-throughput biosensors to quantify protein interactions. PMID:22635097

  7. Calorimetric quantification of linked equilibria in cyclodextrin/lipid/detergent mixtures for membrane-protein reconstitution.

    PubMed

    Textor, Martin; Vargas, Carolyn; Keller, Sandro

    2015-04-01

    Reconstitution from detergent micelles into lipid bilayer membranes is a prerequisite for many in vitro studies on purified membrane proteins. Complexation by cyclodextrins offers an efficient and tightly controllable way of removing detergents for membrane-protein reconstitution, since cyclodextrins sequester detergents at defined stoichiometries and with tuneable affinities. To fully exploit the potential advantages of cyclodextrin for membrane-protein reconstitution, we establish a quantitative model for predicting the supramolecular transition from mixed micelles to vesicles during cyclodextrin-mediated detergent extraction. The model is based on a set of linked equilibria among all pseudophases present in the course of the reconstitution process. Various isothermal titration-calorimetric protocols are used for quantifying a detergent's self-association as well as its colloidal and stoichiometric interactions with lipid and cyclodextrin, respectively. The detergent's critical micellar concentration, the phase boundaries in the lipid/detergent phase diagram, and the dissociation constant of the cyclodextrin/detergent complex thus obtained provide all thermodynamic parameters necessary for a quantitative prediction of the transition from micelles to bilayer membranes during cyclodextrin-driven reconstitution. This is exemplified and validated by stepwise complexation of the detergent lauryldimethylamine N-oxide in mixtures with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine upon titration with 2-hydroxypropyl-β-cyclodextrin, both in the presence and in the absence of the membrane protein Mistic. The calorimetric approach presented herein quantitatively predicts the onset and completion of the reconstitution process, thus obviating cumbersome trial-and-error efforts and facilitating the rational optimisation of reconstitution protocols, and can be adapted to different cyclodextrin/lipid/detergent combinations.

  8. Calorimetric Quantification of Cyclodextrin-Mediated Detergent Extraction for Membrane-Protein Reconstitution.

    PubMed

    Textor, Martin; Keller, Sandro

    2016-01-01

    For many in vitro studies, purified membrane proteins need to be reconstituted from detergent micelles into lipid bilayers to regain their native structures and functions. Stoichiometric complexation of detergent by cyclodextrin provides a tightly controllable strategy for detergent extraction. Here, we describe a practical approach making use of isothermal titration calorimetry to obtain a complete set of thermodynamic parameters that allows for quantitative prediction of the transition from micelles to bilayer membranes during reconstitution. These parameters include the dissociation constant of the cyclodextrin/detergent inclusion complex, the critical micellar concentration of the detergent, and the phase boundaries of the lipid/detergent phase diagram. The underlying theoretical framework involves linked equilibria among all pseudophases, as described previously (Textor, Vargas, & Keller, 2015). This chapter focuses on practical aspects of the approach and discusses caveats and calorimetry-specific details of data analysis. With the entire parameter set at hand, exploration of different reconstitution trajectories within the lipid/detergent phase diagram is possible. Together with the straightforward control over the rate of detergent extraction offered by cyclodextrin complexation, this opens the possibility of systematically tuning and optimizing the reconstitution process of membrane proteins. Provided some particular precautions are taken, the approach can be adapted to many other combinations of proteins, lipids, detergents, and cyclodextrins.

  9. Quantification of Compactness and Local Order in the Ensemble of the Intrinsically Disordered Protein FCP1.

    PubMed

    Gibbs, Eric B; Showalter, Scott A

    2016-09-01

    Intrinsically disordered protein regions (IDRs) partially or completely lack a cooperatively folded structure under native conditions, preventing their equilibrium state from being adequately described by a single structural model. As a direct consequence of their disorder, remarkably few experimental studies have quantified the ensembles IDRs adopt in solution. Here, we conduct unbiased computer simulations of the RAP74 interaction motif from the human phosphatase FCP1 in the unbound state, which provides an ensemble in quantitative agreement with both experimental NMR chemical shift information and small-angle X-ray scattering data. The partially α-helical short linear motif found in the C-terminus of FCP1 has been the subject of extensive biophysical characterization aimed at developing a molecular description for the mechanism of coupled folding and binding and establishing the functional relevance of partial order in the unbound state. The analysis presented here yields a remarkably consistent molecular picture enumerating the diversity of structures present in a "partially formed" helix. Specific interactions, including anticorrelations in backbone dihedral angle fluctuations as well as the transient formation of a helix-stabilizing salt bridge, stabilize the preformed structure in the unbound state. The general consequences of these findings for mechanistic analysis of protein-protein interactions are discussed. PMID:27551949

  10. Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins.

    PubMed

    Yan, Qi; Schmidt, Brigitte F; Perkins, Lydia A; Naganbabu, Matharishwan; Saurabh, Saumya; Andreko, Susan K; Bruchez, Marcel P

    2015-02-21

    Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors. Fluorescence microscopy and flow cytometry demonstrate that this dye does not cross the plasma membrane, binds with high affinity to a dL5** FAP-GPCR fusion construct, activating tagged surface receptors within seconds of addition. The ability to rapidly and selectively label cell surface receptors with a fluorogenic genetically encoded tag allows quantitative imaging and analysis of highly dynamic processes like receptor endocytosis and recycling. PMID:25520058

  11. PSSP-RFE: Accurate Prediction of Protein Structural Class by Recursive Feature Extraction from PSI-BLAST Profile, Physical-Chemical Property and Functional Annotations

    PubMed Central

    Yu, Sanjiu; Zhang, Yuan; Luo, Zhong; Yang, Hua; Zhou, Yue; Zheng, Xiaoqi

    2014-01-01

    Protein structure prediction is critical to functional annotation of the massively accumulated biological sequences, which prompts an imperative need for the development of high-throughput technologies. As a first and key step in protein structure prediction, protein structural class prediction becomes an increasingly challenging task. Amongst most homological-based approaches, the accuracies of protein structural class prediction are sufficiently high for high similarity datasets, but still far from being satisfactory for low similarity datasets, i.e., below 40% in pairwise sequence similarity. Therefore, we present a novel method for accurate and reliable protein structural class prediction for both high and low similarity datasets. This method is based on Support Vector Machine (SVM) in conjunction with integrated features from position-specific score matrix (PSSM), PROFEAT and Gene Ontology (GO). A feature selection approach, SVM-RFE, is also used to rank the integrated feature vectors through recursively removing the feature with the lowest ranking score. The definitive top features selected by SVM-RFE are input into the SVM engines to predict the structural class of a query protein. To validate our method, jackknife tests were applied to seven widely used benchmark datasets, reaching overall accuracies between 84.61% and 99.79%, which are significantly higher than those achieved by state-of-the-art tools. These results suggest that our method could serve as an accurate and cost-effective alternative to existing methods in protein structural classification, especially for low similarity datasets. PMID:24675610

  12. Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers.

    PubMed

    Carlsson, Nils; Borde, Annika; Wölfel, Sebastian; Kerman, Björn; Larsson, Anette

    2011-04-01

    We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595nm) can be identified and corrected by recording absorption spectra in the region of 350-850mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595nm by measuring the sample absorbance at 850nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.

  13. Optimization of an HP Scanjet for quantification of protein electrophoresis gels.

    PubMed

    Kendrick, N C; Johansen, J J; Lee, P R; Santek, D A

    1994-06-01

    An inexpensive desktop scanner, the Hewlett-Packard Scanjet IIp (HP), has been optimized for analysis of protein electrophoresis gels by comparison with a calibrated laser densitometer (Laser). Images from both densitometers were transferred to a personal computer and analyzed with QGEL software. Without correction the HP response was often in poor agreement with the Laser. However, when the HP response to Coomassie blue stained gels and x-ray films was linearized using a HP software option called Emphasis, the HP results agreed with results from the Laser. For 2D gels scanned with appropriate Emphasis applied, spot integrated density values were a constant multiple of 1.8 +/- 0.3 times the corresponding Laser value for x-ray films (CV = 17%) and 2.1 +/- 0.5 for Coomassie blue stained gels (CV = 24%). The highest error was observed for density extremes. For proteins quantified relative to standards using sodium dodecyl sulfate-slab gel electrophoresis, the HP values were within 15% of the Laser values. Data is shown concerning linearity and reproducibility of response, optical density range (about 0 to 1.8 OD units), variability of the imaging field, and resolution of the HP.

  14. Quantification of Endogenous Retinoids

    PubMed Central

    Kane, Maureen A.; Napoli, Joseph L.

    2014-01-01

    Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimer’s disease, type 2 diabetes, obesity, and cancer. PMID:20552420

  15. In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

    PubMed

    Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

  16. Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Reska, M; Ochsmann, E; Kraus, T; Schettgen, T

    2010-08-01

    Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-L-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-L-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography-tandem mass spectrometry method using (13)C(6)-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 microg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 microg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 microg/L (less than 0.3 to 1.1 microg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 microg/L (less than 0.3 to 2.8 microg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.

  17. Quantitative comparison of CrkL-SH3 binding proteins from embryonic murine brain and liver: Implications for developmental signaling and the quantification of protein species variants in bottom-up proteomics.

    PubMed

    Cheerathodi, Mujeeburahim; Vincent, James J; Ballif, Bryan A

    2015-07-01

    A major aim of proteomics is to comprehensively identify and quantify all protein species variants from a given biological source. However, in spite of its tremendous utility, bottom-up proteomic strategies can do little to provide true quantification of distinct whole protein species variants given its reliance on proteolysis. This is particularly true when molecular size information is lost as in gel-free proteomics. Crk and CrkL comprise a family of adaptor proteins that couple upstream phosphotyrosine signals to downstream effectors by virtue of their SH2 and SH3 domains respectively. Here we compare the identification and quantification of CrkL-SH3 binding partners between embryonic murine brain and liver. We also uncover and quantify tissue-specific variants in CrkL-SH3 binding proteins.

  18. An accurate binding interaction model in de novo computational protein design of interactions: if you build it, they will bind.

    PubMed

    London, Nir; Ambroggio, Xavier

    2014-02-01

    Computational protein design efforts aim to create novel proteins and functions in an automated manner and, in the process, these efforts shed light on the factors shaping natural proteins. The focus of these efforts has progressed from the interior of proteins to their surface and the design of functions, such as binding or catalysis. Here we examine progress in the development of robust methods for the computational design of non-natural interactions between proteins and molecular targets such as other proteins or small molecules. This problem is referred to as the de novo computational design of interactions. Recent successful efforts in de novo enzyme design and the de novo design of protein-protein interactions open a path towards solving this problem. We examine the common themes in these efforts, and review recent studies aimed at understanding the nature of successes and failures in the de novo computational design of interactions. While several approaches culminated in success, the use of a well-defined structural model for a specific binding interaction in particular has emerged as a key strategy for a successful design, and is therefore reviewed with special consideration.

  19. Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein

    PubMed Central

    Yang, Ting; Arslanova, Dilyara; Gu, Yongli; Augelli-Szafran, Corinne; Xia, Weiming

    2008-01-01

    Background Deposition of amyloid-β protein (Aβ) is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from γ-secretase cleavage of amyloid precursor protein (APP). In addition to APP, γ-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development. Results To explore selective γ-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the γ-secretase cleavage of APP and Notch. When two potent γ-secretase inhibitors, compound E (cpd E) and DAPT, were used in a conventional in vitro γ-secretase activity assay, cpd E completely blocked Aβ generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate NotchΔE. Both cpd E and DAPT were more potent in blocking Aβ generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H) binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Aβ-like" (Nβ*) peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nβ* peptides by ELISA confirmed that EC50's of cpd E were much higher for Nβ* than Aβ. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish. Conclusion Our ELISA-based quantification of Aβ and Nβ* in combination with the test in zebrafish provides a novel

  20. aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events.

    PubMed

    Harrison, Thomas; Ruiz, Jaime; Sloan, Daniel B; Ben-Hur, Asa; Boucher, Christina

    2016-01-01

    Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The [Formula: see text] class contains tandem [Formula: see text]-type motif sequences, and the [Formula: see text] class contains alternating [Formula: see text], [Formula: see text] and [Formula: see text] type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a [Formula: see text]-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the [Formula: see text] class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for [Formula: see text]-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve. PMID:27560805

  1. aPPRove: An HMM-Based Method for Accurate Prediction of RNA-Pentatricopeptide Repeat Protein Binding Events

    PubMed Central

    Harrison, Thomas; Ruiz, Jaime; Sloan, Daniel B.; Ben-Hur, Asa; Boucher, Christina

    2016-01-01

    Pentatricopeptide repeat containing proteins (PPRs) bind to RNA transcripts originating from mitochondria and plastids. There are two classes of PPR proteins. The P class contains tandem P-type motif sequences, and the PLS class contains alternating P, L and S type sequences. In this paper, we describe a novel tool that predicts PPR-RNA interaction; specifically, our method, which we call aPPRove, determines where and how a PLS-class PPR protein will bind to RNA when given a PPR and one or more RNA transcripts by using a combinatorial binding code for site specificity proposed by Barkan et al. Our results demonstrate that aPPRove successfully locates how and where a PPR protein belonging to the PLS class can bind to RNA. For each binding event it outputs the binding site, the amino-acid-nucleotide interaction, and its statistical significance. Furthermore, we show that our method can be used to predict binding events for PLS-class proteins using a known edit site and the statistical significance of aligning the PPR protein to that site. In particular, we use our method to make a conjecture regarding an interaction between CLB19 and the second intronic region of ycf3. The aPPRove web server can be found at www.cs.colostate.edu/~approve. PMID:27560805

  2. Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration.

    PubMed

    Guermant, C; Azarkan, M; Smolders, N; Baeyens-Volant, D; Nijs, M; Paul, C; Brygier, J; Vincentelli, J; Looze, Y

    2000-01-01

    Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay. PMID:10610688

  3. Quantification of a biomarker of acetaminophen protein adducts in human serum by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: clinical and animal model applications.

    PubMed

    Cook, Sarah F; King, Amber D; Chang, Yan; Murray, Gordon J; Norris, Hye-Ryun K; Dart, Richard C; Green, Jody L; Curry, Steven C; Rollins, Douglas E; Wilkins, Diana G

    2015-03-15

    The aims of this study were to develop, validate, and apply a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantification of protein-derived 3-(cystein-S-yl)-acetaminophen (APAP-Cys) in human serum. Formation of acetaminophen (APAP) protein adducts is thought to be a critical, early event in the development of APAP-induced hepatotoxicity, and quantification of these protein adducts in human serum represents a valuable tool for assessment of APAP exposure, metabolism, and toxicity. In the reported procedure, serum samples were first dialyzed or passed through gel filtration columns to remove APAP-Cys not covalently bound to proteins. Serum eluates were then subjected to enzymatic protease digestion to liberate protein-bound APAP-Cys. Norbuprenorphine-D3 was utilized as an internal standard (IS). APAP-Cys and IS were recovered from digested serum by protein precipitation with acetonitrile, and sample extracts were analyzed by HPLC-ESI-MS/MS. The method was validated by assessment of intra- and inter-assay accuracy and imprecision on two different analytical instrument platforms. APAP-Cys could be accurately quantified from 0.010 to 10μM, and intra- and inter-assay imprecision were <15% on both analytical instruments. APAP-Cys was stable in human serum for three freeze-thaw cycles and for 24h at ambient temperature. Extracted samples were stable when stored in refrigerated autosamplers for the typical duration of analysis or when stored at -20°C for six days. Results from process efficiency and matrix effect experiments indicated adequate recovery from human serum and insignificant ion suppression or enhancement. The utility and sensitivity of the reported procedure were illustrated by analysis of clinical samples collected from subjects taking chronic, therapeutic doses of APAP. Applicability to other biological matrices was also demonstrated by measurement of protein-derived APAP-Cys in plasma

  4. HubAlign: an accurate and efficient method for global alignment of protein–protein interaction networks

    PubMed Central

    Hashemifar, Somaye; Xu, Jinbo

    2014-01-01

    Motivation: High-throughput experimental techniques have produced a large amount of protein–protein interaction (PPI) data. The study of PPI networks, such as comparative analysis, shall benefit the understanding of life process and diseases at the molecular level. One way of comparative analysis is to align PPI networks to identify conserved or species-specific subnetwork motifs. A few methods have been developed for global PPI network alignment, but it still remains challenging in terms of both accuracy and efficiency. Results: This paper presents a novel global network alignment algorithm, denoted as HubAlign, that makes use of both network topology and sequence homology information, based upon the observation that topologically important proteins in a PPI network usually are much more conserved and thus, more likely to be aligned. HubAlign uses a minimum-degree heuristic algorithm to estimate the topological and functional importance of a protein from the global network topology information. Then HubAlign aligns topologically important proteins first and gradually extends the alignment to the whole network. Extensive tests indicate that HubAlign greatly outperforms several popular methods in terms of both accuracy and efficiency, especially in detecting functionally similar proteins. Availability: HubAlign is available freely for non-commercial purposes at http://ttic.uchicago.edu/∼hashemifar/software/HubAlign.zip Contact: jinboxu@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161231

  5. Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities.

    PubMed

    Venner, Eric; Lisewski, Andreas Martin; Erdin, Serkan; Ward, R Matthew; Amin, Shivas R; Lichtarge, Olivier

    2010-01-01

    High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC) levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.

  6. freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis.

    PubMed

    Deng, Ning; Li, Zhenye; Pan, Chao; Duan, Huilong

    2015-01-01

    Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

  7. A highly accurate protein structural class prediction approach using auto cross covariance transformation and recursive feature elimination.

    PubMed

    Li, Xiaowei; Liu, Taigang; Tao, Peiying; Wang, Chunhua; Chen, Lanming

    2015-12-01

    Structural class characterizes the overall folding type of a protein or its domain. Many methods have been proposed to improve the prediction accuracy of protein structural class in recent years, but it is still a challenge for the low-similarity sequences. In this study, we introduce a feature extraction technique based on auto cross covariance (ACC) transformation of position-specific score matrix (PSSM) to represent a protein sequence. Then support vector machine-recursive feature elimination (SVM-RFE) is adopted to select top K features according to their importance and these features are input to a support vector machine (SVM) to conduct the prediction. Performance evaluation of the proposed method is performed using the jackknife test on three low-similarity datasets, i.e., D640, 1189 and 25PDB. By means of this method, the overall accuracies of 97.2%, 96.2%, and 93.3% are achieved on these three datasets, which are higher than those of most existing methods. This suggests that the proposed method could serve as a very cost-effective tool for predicting protein structural class especially for low-similarity datasets.

  8. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases

    PubMed Central

    Floden, Evan W.; Tommaso, Paolo D.; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-01-01

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. PMID:27106060

  9. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

    PubMed

    Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-07-01

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee.

  10. Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of deltaC(p) of protein unfolding.

    PubMed

    Szyperski, Thomas; Mills, Jeffrey L; Perl, Dieter; Balbach, Jochen

    2006-04-01

    Cold and heat denaturation of the double mutant Arg 3-->Glu/Leu 66-->Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D (1)H NMR spectroscopy in the temperature range from -12 degrees C in supercooled water up to +70 degrees C. The fraction of unfolded protein, f (u), was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be -11 degrees C and 39 degrees C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, DeltaC(p) of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.

  11. Advances in inline quantification of co-eluting proteins in chromatography: Process-data-based model calibration and application towards real-life separation issues.

    PubMed

    Brestrich, Nina; Sanden, Adrian; Kraft, Axel; McCann, Karl; Bertolini, Joseph; Hubbuch, Jürgen

    2015-07-01

    Pooling decisions in preparative liquid chromatography for protein purification are usually based on univariate UV absorption measurements that are not able to differentiate between product and co-eluting contaminants. This can result in inconsistent pool purities or yields, if there is a batch-to-batch variability of the feedstock. To overcome this analytical bottleneck, a tool for selective inline quantification of co-eluting model proteins using mid-UV absorption spectra and Partial Least Squares Regression (PLS) was presented in a previous study and applied for real-time pooling decisions. In this paper, a process-data-based method for the PLS model calibration will be introduced that allows the application of the tool towards chromatography steps of real-life processes. The process-data-based calibration method uses recorded inline mid-UV absorption spectra that are correlated with offline fraction analytics to calibrate PLS models. In order to generate average spectra from the inline data, a Visual Basic for Application macro was successfully developed. The process-data-based model calibration was established using a ternary model protein system. Afterwards, it was successfully demonstrated in two case studies that the calibration method is applicable towards real-life separation issues. The calibrated PLS models allowed a successful quantification of the co-eluting species in a cation-exchange-based aggregate and fraction removal during the purification of monoclonal antibodies and of co-eluting serum proteins in an anion-exchange-based purification of Cohn supernatant I. Consequently, the presented process-data-based PLS model calibration in combination with the tool for selective inline quantification has a great potential for the monitoring of future chromatography steps and may contribute to manage batch-to-batch variability by real-time pooling decisions. PMID:25683378

  12. 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

    2014-12-01

    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. PMID:25403019

  13. Advances in inline quantification of co-eluting proteins in chromatography: Process-data-based model calibration and application towards real-life separation issues.

    PubMed

    Brestrich, Nina; Sanden, Adrian; Kraft, Axel; McCann, Karl; Bertolini, Joseph; Hubbuch, Jürgen

    2015-07-01

    Pooling decisions in preparative liquid chromatography for protein purification are usually based on univariate UV absorption measurements that are not able to differentiate between product and co-eluting contaminants. This can result in inconsistent pool purities or yields, if there is a batch-to-batch variability of the feedstock. To overcome this analytical bottleneck, a tool for selective inline quantification of co-eluting model proteins using mid-UV absorption spectra and Partial Least Squares Regression (PLS) was presented in a previous study and applied for real-time pooling decisions. In this paper, a process-data-based method for the PLS model calibration will be introduced that allows the application of the tool towards chromatography steps of real-life processes. The process-data-based calibration method uses recorded inline mid-UV absorption spectra that are correlated with offline fraction analytics to calibrate PLS models. In order to generate average spectra from the inline data, a Visual Basic for Application macro was successfully developed. The process-data-based model calibration was established using a ternary model protein system. Afterwards, it was successfully demonstrated in two case studies that the calibration method is applicable towards real-life separation issues. The calibrated PLS models allowed a successful quantification of the co-eluting species in a cation-exchange-based aggregate and fraction removal during the purification of monoclonal antibodies and of co-eluting serum proteins in an anion-exchange-based purification of Cohn supernatant I. Consequently, the presented process-data-based PLS model calibration in combination with the tool for selective inline quantification has a great potential for the monitoring of future chromatography steps and may contribute to manage batch-to-batch variability by real-time pooling decisions.

  14. 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

    2014-12-01

    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.

  15. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

    PubMed

    Heudi, Olivier; Barteau, Samuel; Zimmer, Dieter; Schmidt, Joerg; Bill, Kurt; Lehmann, Natalie; Bauer, Christian; Kretz, Olivier

    2008-06-01

    Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC

  16. Custom 4-Plex DiLeu Isobaric Labels Enable Relative Quantification of Urinary Proteins in Men with Lower Urinary Tract Symptoms (LUTS)

    PubMed Central

    Greer, Tyler; Hao, Ling; Nechyporenko, Anatoliy; Lee, Sanghee; Vezina, Chad M.; Ricke, Will A.; Marker, Paul C.; Bjorling, Dale E.; Bushman, Wade; Li, Lingjun

    2015-01-01

    The relative quantification of proteins using liquid chromatography mass spectrometry (LC-MS) has allowed researchers to compile lists of potential disease markers. These complex quantitative workflows often include isobaric labeling of enzymatically-produced peptides to analyze their relative abundances across multiple samples in a single LC-MS run. Recent efforts by our lab have provided scientists with cost-effective alternatives to expensive commercial labels. Although the quantitative performance of these dimethyl leucine (DiLeu) labels has been reported using known ratios of complex protein and peptide standards, their potential in large-scale proteomics studies using a clinically relevant system has never been investigated. Our work rectifies this oversight by implementing 4-plex DiLeu to quantify proteins in the urine of aging human males who suffer from lower urinary tract symptoms (LUTS). Protein abundances in 25 LUTS and 15 control patients were compared, revealing that of the 836 proteins quantified, 50 were found to be differentially expressed (>20% change) and statistically significant (p-value <0.05). Gene ontology (GO) analysis of the differentiated proteins showed that many were involved in inflammatory responses and implicated in fibrosis. While confirmation of individual protein abundance changes would be required to verify protein expression, this study represents the first report using the custom isobaric label, 4-plex DiLeu, to quantify protein abundances in a clinically relevant system. PMID:26267142

  17. Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

    PubMed

    Vlaming, M L H; van Duijn, E; Dillingh, M R; Brands, R; Windhorst, A D; Hendrikse, N H; Bosgra, S; Burggraaf, J; de Koning, M C; Fidder, A; Mocking, J A J; Sandman, H; de Ligt, R A F; Fabriek, B O; Pasman, W J; Seinen, W; Alves, T; Carrondo, M; Peixoto, C; Peeters, P A M; Vaes, W H J

    2015-08-01

    Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 μg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs. PMID:25869840

  18. Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0

    NASA Astrophysics Data System (ADS)

    The, Matthew; MacCoss, Michael J.; Noble, William S.; Käll, Lukas

    2016-08-01

    Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license.

  19. Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0

    NASA Astrophysics Data System (ADS)

    The, Matthew; MacCoss, Michael J.; Noble, William S.; Käll, Lukas

    2016-11-01

    Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license.

  20. Large-format imaging plate and weissenberg camera for accurate protein crystallographic data collection using synchrotron radiation.

    PubMed

    Sakabe, K; Sasaki, K; Watanabe, N; Suzuki, M; Wang, Z G; Miyahara, J; Sakabe, N

    1997-05-01

    Off-line and on-line protein data-collection systems using an imaging plate as a detector are described and their components reported. The off-line scanner IPR4080 was developed for a large-format imaging plate ;BASIII' of dimensions 400 x 400 mm and 400 x 800 mm. The characteristics of this scanner are a dynamic range of 10(5) photons pixel(-1), low background noise and high sensitivity. A means of reducing electronic noise and a method for finding the origin of the noise are discussed in detail. A dedicated screenless Weissenberg camera matching IPR4080 with synchrotron radiation was developed and installed on beamline BL6B at the Photon Factory. This camera can attach one or two sheets of 400 x 800 mm large-format imaging plate inside the film cassette by evacuation. The positional reproducibility of the imaging plate on the cassette is so good that the data can be processed by batch job. Data of 93% completeness up to 1.6 A resolution were collected on a single axis rotation and the value of R(merge) becomes 4% from a tetragonal lysozyme crystal using a set of two imaging-plate sheets. Comparing two types of imaging plates, the signal-to-noise ratio of the ST-VIP-type imaging plate is 25% better than that of the BASIII-type imaging plate for protein data collection using 1.0 and 0.7 A X-rays. A new on-line protein data-collection system with imaging plates is specially designed to use synchrotron radiation X-rays at maximum efficiency.

  1. Quantification of viral proteins of the avian H7 subtype of influenza virus: an isotope dilution mass spectrometry method applicable for producing more rapid vaccines in the case of an influenza pandemic.

    PubMed

    Santana, Wanda I; Williams, Tracie L; Winne, Emily K; Pirkle, James L; Barr, John R

    2014-05-01

    Vaccination is the most effective means to prevent influenza and its serious complications. Influenza viral strains undergo rapid mutations of the surface proteins hemagglutinin (HA) and neuraminidase (NA) requiring vaccines to be frequently updated to include current circulating strains. It is nearly impossible to predict which strains will be circulating in the next influenza season. It is, therefore, imperative that the process of producing a vaccine be streamlined and as swift as possible. We have developed an isotope dilution mass spectrometry (IDMS) method to quantify HA and NA in H7N7, H7N2, and H7N9 influenza. The IDMS method involves enzymatic digestion of viral proteins and the specific detection of evolutionarily conserved target peptides. The four target peptides that were initially chosen for analysis of the HA protein of H7N2 and H7N7 subtypes were conserved and available for analysis of the H7N9 subtype that circulated in China in the spring of 2013. Thus, rapid response to the potential pandemic was realized. Quantification of a protein is performed by employing multiple peptides to ensure that the enzymatic digestion of the protein is efficient in the region of the target peptides, verify the accuracy of the measurement, and provide flexibility in the case of amino acid changes among newly emerging strains. The IDMS method is an accurate, sensitive, and selective method to quantify the amount of HA and NA antigens in primary liquid standards, crude allantoic fluid, purified virus samples, and final vaccine presentations.

  2. The proteomics quantification dilemma.

    PubMed

    Jungblut, Peter R

    2014-07-31

    Proteomics is dominated today by the protein expression discourse, which favorites the bottom-up approach because of its high throughput and its high sensitivity. For quantification this proceeding is misleading, if a protein is present with more than one protein species in the sample to be analyzed. The protein speciation discourse considers this more realistic situation and affords the top-down procedures or at least a separation of the protein species in advance to identification and quantification. Today all of the top-down procedures are one order of magnitude less sensitive than the bottom-up ones. To increase sensitivity and to increase throughput are major challenges for proteomics of the next years. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez. PMID:24681132

  3. MetaboQuant: a tool combining individual peak calibration and outlier detection for accurate metabolite quantification in 1D (1)H and (1)H-(13)C HSQC NMR spectra.

    PubMed

    Klein, Matthias S; Oefner, Peter J; Gronwald, Wolfram

    2013-05-01

    Solution nuclear magnetic resonance (NMR) spectroscopy is widely used to analyze complex mixtures of organic compounds such as biological fluids and tissue extracts. Targeted profiling approaches with reliable compound quantitifcation are hampered, however, by signal overlap and other interferences. Here, we present a tool named MetaboQuant for automated compound quantification from pre-processed 1D and 2D heteronuclear single quantum coherence (HSQC) NMR spectral data and concomitant validation of results. Performance of MetaboQuant was tested on a urinary spike-in data set and compared with other quantification strategies. The use of individual calibration factors in combination with the validation algorithms of MetaboQuant raises the reliability of the quantification results. MetaboQuant can be downloaded at http://genomics.uni-regensburg.de/site/institute/software/metaboquant/ as stand-alone software for Windows or run on other operating systems from within Matlab. Separate software for peak fitting and integration is necessary in order to use MetaboQuant. PMID:23662895

  4. Quantification and characterization of maize lipid transfer protein, a food allergen, by liquid chromatography with ultraviolet and mass spectrometric detection.

    PubMed

    Kuppannan, Krishna; Albers, David R; Schafer, Barry W; Dielman, Demetrius; Young, Scott A

    2011-01-15

    Maize (Zea mays) is not considered a major allergenic food; however, when food induced allergenic and immunologic reactions have been implicated to maize, lipid transfer proteins (LTPs) have been identified as major allergens. LTP is an extremely stable protein that is resistant to both proteolytic attack and food processing, which permits the allergen to reach the gastrointestinal immune system in an immunogenic and allergenic conformation, allowing sensitization and induction of systemic symptoms. They are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting severe symptoms. We have purified and characterized an endogenous ~9 kDa LTP from maize kernels. The maize LTP consists of 93 amino acid residues and has a M(r) of 9046.1 Da, determined by electrospray ionization mass spectrometry. Following accurate identification and characterization of maize LTP, a highly specific and quantitative assay using liquid chromatography with ultraviolet and mass spectrometric detection was developed. The present assay enables determination of LTP over a concentration range from 29 to 1030 μg/g in maize kernel samples. Assay recovery (percent relative error, % RE) was measured at 11 different concentrations ranging from 4 to 147 μg/mL and did not exceed 5.1%. The precision (percent coefficient of variation, % CV) was measured at 3 concentrations on each of 4 days and did not exceed 14.4%. The method was applied to evaluate the levels of LTP in 14 different maize lines. To our knowledge, this represents the first quantitative liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS) assay for the determination of LTP for the assessment of a food allergen.

  5. Application of an LC-MS/MS method for the simultaneous quantification of human intestinal transporter proteins absolute abundance using a QconCAT technique.

    PubMed

    Harwood, M D; Achour, B; Russell, M R; Carlson, G L; Warhurst, G; Rostami-Hodjegan, A

    2015-06-10

    Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to

  6. Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

    SciTech Connect

    Nguyen, Hung T.; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois; Case, David A.

    2014-12-14

    A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb{sup +} and Sr{sup 2+}) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein–Zernike equations, with results from the Kovalenko–Hirata closure being closest to experiment for the cases studied here.

  7. Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids.

    PubMed

    Nguyen, Hung T; Pabit, Suzette A; Meisburger, Steve P; Pollack, Lois; Case, David A

    2014-12-14

    A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb(+) and Sr(2+)) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein-Zernike equations, with results from the Kovalenko-Hirata closure being closest to experiment for the cases studied here.

  8. Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking.

    PubMed

    Ogura, Toshihiko; Iwasaki, Kenji; Sato, Chikara

    2003-09-01

    In single-particle analysis, a three-dimensional (3-D) structure of a protein is constructed using electron microscopy (EM). As these images are very noisy in general, the primary process of this 3-D reconstruction is the classification of images according to their Euler angles, the images in each classified group then being averaged to reduce the noise level. In our newly developed strategy of classification, we introduce a topology representing network (TRN) method. It is a modified method of a growing neural gas network (GNG). In this system, a network structure is automatically determined in response to the images input through a growing process. After learning without a masking procedure, the GNG creates clear averages of the inputs as unit coordinates in multi-dimensional space, which are then utilized for classification. In the process, connections are automatically created between highly related units and their positions are shifted where the inputs are distributed in multi-dimensional space. Consequently, several separated groups of connected units are formed. Although the interrelationship of units in this space are not easily understood, we succeeded in solving this problem by converting the unit positions into two-dimensional (2-D) space, and by further optimizing the unit positions with the simulated annealing (SA) method. In the optimized 2-D map, visualization of the connections of units provided rich information about clustering. As demonstrated here, this method is clearly superior to both the multi-variate statistical analysis (MSA) and the self-organizing map (SOM) as a classification method and provides a first reliable classification method which can be used without masking for very noisy images. PMID:14572474

  9. Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

    NASA Astrophysics Data System (ADS)

    Nguyen, Hung T.; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois; Case, David A.

    2014-12-01

    A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb+ and Sr2+) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein-Zernike equations, with results from the Kovalenko-Hirata closure being closest to experiment for the cases studied here.

  10. Direct quantification of protein partitioning in oil-in-water emulsion by front-face fluorescence: avoiding the need for centrifugation.

    PubMed

    Granger, C; Barey, P; Toutain, J; Cansell, M

    2005-07-10

    The quantification of proteins adsorbed at the oil-in-water interface is often difficult since it requires separation of fat globules from the aqueous phase that may damage the fat globule size and/or modify the interfacial composition. Front-face fluorescence spectroscopy was used to characterize the protein partitioning between the aqueous and oil phases of emulsions without separating these two phases. Different emulsions based on skim milk powder (SMP), two mono- and di-glyceride (MDG) mixtures (saturated and partially unsaturated), and three fats (hydrogenated and refined coconut oils and refined palm oil) were studied. The impact of an ageing period (24 h at 4 degrees C) was also investigated to typify the first step of ice cream processing. The emulsions were characterized for protein partitioning, immediately following emulsification and after ageing, using the Bradford spectrophotometric method, applied to the aqueous phase recovered after emulsion centrifugation. In parallel, the emulsions were characterized by their tryptophan emission fluorescence spectra. The area of the peaks at 333 nm, of the fourth-derivative fluorescence spectra corresponding to the amount of proteins present in the aqueous phase of emulsions, was well correlated with the Bradford measurements (r2=0.91). This amount was also calculated from the fluorescence calibration curve obtained with SMP in solution. In conclusion, front-face fluorescence spectroscopy appeared to be a powerful and simple technique allowing the quantification of different populations of protein in an emulsified system, i.e., in the aqueous phase and loaded at the fat globule interface. PMID:15946827

  11. Accurate flexible fitting of high-resolution protein structures to small-angle x-ray scattering data using a coarse-grained model with implicit hydration shell.

    PubMed

    Zheng, Wenjun; Tekpinar, Mustafa

    2011-12-21

    Small-angle x-ray scattering (SAXS) is a powerful technique widely used to explore conformational states and transitions of biomolecular assemblies in solution. For accurate model reconstruction from SAXS data, one promising approach is to flexibly fit a known high-resolution protein structure to low-resolution SAXS data by computer simulations. This is a highly challenging task due to low information content in SAXS data. To meet this challenge, we have developed what we believe to be a novel method based on a coarse-grained (one-bead-per-residue) protein representation and a modified form of the elastic network model that allows large-scale conformational changes while maintaining pseudobonds and secondary structures. Our method optimizes a pseudoenergy that combines the modified elastic-network model energy with a SAXS-fitting score and a collision energy that penalizes steric collisions. Our method uses what we consider a new implicit hydration shell model that accounts for the contribution of hydration shell to SAXS data accurately without explicitly adding waters to the system. We have rigorously validated our method using five test cases with simulated SAXS data and three test cases with experimental SAXS data. Our method has successfully generated high-quality structural models with root mean-squared deviation of 1 ∼ 3 Å from the target structures.

  12. How Iron-Containing Proteins Control Dioxygen Chemistry: A Detailed Atomic Level Description Via Accurate Quantum Chemical and Mixed Quantum Mechanics/Molecular Mechanics Calculations.

    SciTech Connect

    Friesner, Richard A.; Baik, Mu-Hyun; Gherman, Benjamin F.; Guallar, Victor; Wirstam, Maria E.; Murphy, Robert B.; Lippard, Stephen J.

    2003-03-01

    Over the past several years, rapid advances in computational hardware, quantum chemical methods, and mixed quantum mechanics/molecular mechanics (QM/MM) techniques have made it possible to model accurately the interaction of ligands with metal-containing proteins at an atomic level of detail. In this paper, we describe the application of our computational methodology, based on density functional (DFT) quantum chemical methods, to two diiron-containing proteins that interact with dioxygen: methane monooxygenase (MMO) and hemerythrin (Hr). Although the active sites are structurally related, the biological function differs substantially. MMO is an enzyme found in methanotrophic bacteria and hydroxylates aliphatic C-H bonds, whereas Hr is a carrier protein for dioxygen used by a number of marine invertebrates. Quantitative descriptions of the structures and energetics of key intermediates and transition states involved in the reaction with dioxygen are provided, allowing their mechanisms to be compared and contrasted in detail. An in-depth understanding of how the chemical identity of the first ligand coordination shell, structural features, electrostatic and van der Waals interactions of more distant shells control ligand binding and reactive chemistry is provided, affording a systematic analysis of how iron-containing proteins process dioxygen. Extensive contact with experiment is made in both systems, and a remarkable degree of accuracy and robustness of the calculations is obtained from both a qualitative and quantitative perspective.

  13. Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance.

    PubMed

    Florentinus, Angelica K; Bowden, Peter; Sardana, Girish; Diamandis, Eleftherios P; Marshall, John G

    2012-02-01

    The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.

  14. PredPPCrys: Accurate Prediction of Sequence Cloning, Protein Production, Purification and Crystallization Propensity from Protein Sequences Using Multi-Step Heterogeneous Feature Fusion and Selection

    PubMed Central

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed ‘PredPPCrys’ using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of

  15. Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.

    PubMed

    Rioja, I; Bush, K A; Buckton, J B; Dickson, M C; Life, P F

    2004-07-01

    Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1beta, TNF-alpha and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1beta and IL-6, but not for TNF-alpha, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1beta and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-alpha in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.

  16. Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey.

    PubMed

    Sturaro, Alba; De Marchi, Massimo; Masi, Antonio; Cassandro, Martino

    2016-01-01

    In the dairy industry, membrane filtration is used to reduce the amount of whey waste and, simultaneously, to recover whey proteins (WP). The composition of WP can strongly affect the filtration treatment of whey, and rapid determination of WP fractions would be of interest for dairy producers to monitor WP recovery. This study aimed to develop mid-infrared spectroscopy (MIRS) prediction models for the rapid quantification of protein in sweet whey, using a validated rapid reversed phase (RP)-HPLC as a reference method. Quantified WP included α-lactalbumin (α-LA), β-lactoglobulin (β-LG) A and B, bovine serum albumin, caseinomacropeptides, and proteose peptone. Validation of RP-HPLC was performed by calculating the relative standard deviation (RSD) in repeatability and reproducibility tests for WP retention time and peak areas. Samples of liquid whey (n=187) were analyzed by RP-HPLC and scanned through MIRS to collect spectral information (900 to 4,000 cm(-1)); statistical analysis was carried out through partial least squares regression and random cross-validation procedure. Retention times in RP-HPLC method were stable (RSD between 0.03 and 0.80%), whereas the RSD of peak area (from 0.25 to 8.48%) was affected by WP relative abundance. Higher coefficients of determination in validation for MIRS model were obtained for protein fractions present in whey in large amounts, such as β-LG (0.58), total identified WP (0.58), and α-LA (0.56). Results of this study suggest that MIRS is an easy method for rapid quantification of detail protein in sweet whey, even if better resolution was achieved with the method based on RP-HPLC. The prediction of WP in sweet whey by MIRS might be used for screening and for classifying sweet whey according to its total and individual WP contents.

  17. Development of an immunohistochemical protein quantification system in conjunction with tissue microarray technology for identifying predictive biomarkers for phosphatidylinositol 3-kinase inhibitors.

    PubMed

    Isoyama, Sho; Yoshimi, Hisashi; Dan, Shingo; Okamura, Mutsumi; Seki, Mariko; Irimura, Tatsuro; Yamori, Takao

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials. PMID:22975517

  18. Quantification of proteins using enhanced etching of Ag coated Au nanorods by the Cu2+/bicinchoninic acid pair with improved sensitivity

    NASA Astrophysics Data System (ADS)

    Liu, Wenqi; Hou, Shuai; Yan, Jiao; Zhang, Hui; Ji, Yinglu; Wu, Xiaochun

    2015-12-01

    Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical simulation shows that Au nanorods with large aspect ratios and small core sizes show high detection sensitivity. Via optimized sensor design, we achieved an increased sensitivity (the limit of detection was 3.4 ng ml-1) and a wide working range (0.5 to 1000 μg ml-1) compared with the traditional BCA assay. The universal applicability of our method to various proteins further proves its potential in practical applications.Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical

  19. LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer.

    PubMed

    Veit, Johannes; Sachsenberg, Timo; Chernev, Aleksandar; Aicheler, Fabian; Urlaub, Henning; Kohlbacher, Oliver

    2016-09-01

    Modern mass spectrometry setups used in today's proteomics studies generate vast amounts of raw data, calling for highly efficient data processing and analysis tools. Software for analyzing these data is either monolithic (easy to use, but sometimes too rigid) or workflow-driven (easy to customize, but sometimes complex). Thermo Proteome Discoverer (PD) is a powerful software for workflow-driven data analysis in proteomics which, in our eyes, achieves a good trade-off between flexibility and usability. Here, we present two open-source plugins for PD providing additional functionality: LFQProfiler for label-free quantification of peptides and proteins, and RNP(xl) for UV-induced peptide-RNA cross-linking data analysis. LFQProfiler interacts with existing PD nodes for peptide identification and validation and takes care of the entire quantitative part of the workflow. We show that it performs at least on par with other state-of-the-art software solutions for label-free quantification in a recently published benchmark ( Ramus, C.; J. Proteomics 2016 , 132 , 51 - 62 ). The second workflow, RNP(xl), represents the first software solution to date for identification of peptide-RNA cross-links including automatic localization of the cross-links at amino acid resolution and localization scoring. It comes with a customized integrated cross-link fragment spectrum viewer for convenient manual inspection and validation of the results. PMID:27476824

  20. Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Lüthi, Guillaume; Blangy, Valeria; Eap, Chin B; Ansermot, Nicolas

    2013-04-15

    A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2 μm column (Acquity UPLC BEH C18 1.7 μm, 2.1×50 mm) in 5 min with a gradient of ammonium formate 20 mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1 ng/ml for buprenorphine and 0.25 ng/ml for norbuprenorphine. The upper limit of quantification was 250 ng/ml for both drugs. Trueness (98.4-113.7%), repeatability (1.9-7.7%), intermediate precision (2.6-7.9%) and internal standard-normalized matrix effects (94-101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development. PMID:23357637

  1. Label-free single-cell protein quantification using a drop-based mix-and-read system

    PubMed Central

    Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan A.; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, David A.

    2015-01-01

    Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry. PMID:26234416

  2. A rapid and accurate method for determining protein content in dairy products based on asynchronous-injection alternating merging zone flow-injection spectrophotometry.

    PubMed

    Liang, Qin-Qin; Li, Yong-Sheng

    2013-12-01

    An accurate and rapid method and a system to determine protein content using asynchronous-injection alternating merging zone flow-injection spectrophotometry based on reaction between coomassie brilliant blue G250 (CBBG) and protein was established. Main merit of our approach is that it can avoid interferences of other nitric-compounds in samples, such as melamine and urea. Optimized conditions are as follows: Concentrations of CBBG, polyvinyl alcohol (PVA), NaCl and HCl are 150 mg/l, 30 mg/l, 0.1 mol/l and 1.0% (v/v), respectively; volumes of the sample and reagent are 150 μl and 30 μl, respectively; length of a reaction coil is 200 cm; total flow rate is 2.65 ml/min. The linear range of the method is 0.5-15 mg/l (BSA), its detection limit is 0.05 mg/l, relative standard deviation is less than 1.87% (n=11), and analytical speed is 60 samples per hour.

  3. Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

    PubMed

    Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

    2014-09-01

    Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes. PMID:25333063

  4. Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens.

    PubMed

    Stephan, Oliver; Weisz, Nancy; Vieths, Stefan; Weiser, Tanja; Rabe, Burghard; Vatterott, Wolfgang

    2004-01-01

    In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery- and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.

  5. Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.

    PubMed

    Toledo, José Carlos; Audi, Renata; Ogusucu, Renata; Monteiro, Gisele; Netto, Luis Eduardo Soares; Augusto, Ohara

    2011-05-01

    Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.

  6. Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli.

    PubMed

    Arike, L; Valgepea, K; Peil, L; Nahku, R; Adamberg, K; Vilu, R

    2012-09-18

    Three different label-free proteome quantification methods--APEX, emPAI and iBAQ--were evaluated to measure proteome-wide protein concentrations in the cell. All the methods were applied to a sample from Escherichia coli chemostat culture. A Pearson squared correlation of approximately 0.6 among the three quantification methods was demonstrated. Importantly, the sum of quantified proteins by iBAQ and emPAI corresponded with the Lowry total protein quantification, demonstrating applicability of label-free methods for an accurate calculation of protein concentrations at the proteome level. The iBAQ method showed the best correlation between biological replicates, a normal distribution among all protein abundances, and the lowest variation among ribosomal protein abundances, which are expected to have equal amounts. Absolute quantitative proteome data enabled us to evaluate metabolic cost for protein synthesis and apparent catalytic activities of enzymes by integration with flux analysis. All the methods demonstrated similar ATP costs for protein synthesis for different cellular processes and that costs for expressing biomass synthesis related proteins were higher than those for energy generation. Importantly, catalytic activities of energy metabolism enzymes were an order or two higher than those of monomer synthesis. Interestingly, a staircase-like protein expression was demonstrated for most of the transcription units. PMID:22771841

  7. Long-Gradient Separations Coupled with Selected Reaction Monitoring for Highly Sensitive, Large Scale Targeted Protein Quantification in a Single Analysis

    SciTech Connect

    Shi, Tujin; Fillmore, Thomas L.; Gao, Yuqian; Zhao, Rui; He, Jintang; Schepmoes, Athena A.; Nicora, Carrie D.; Wu, Chaochao; Chambers, Justin L.; Moore, Ronald J.; Kagan, Jacob; Srivastava, Sudhir; Liu, Alvin Y.; Rodland, Karin D.; Liu, Tao; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2013-10-01

    Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM offers at least 3 times higher multiplexing capacity than conventional LC-SRM due to ~3-fold increase in average peak widths for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.

  8. Quantification of Poly(ADP-ribose)-Modified Proteins in Cerebrospinal Fluid from Infants and Children after Traumatic Brain Injury

    PubMed Central

    Fink, Ericka L.; Lai, Yichen; Zhang, Xiaopeng; Janesko-Feldman, Keri; Adelson, P. David; Szabó, Csaba; Berger, Rachel P.; Sarnaik, Ajit A.; Kochanek, Patrick M.; Clark, Robert S. B.

    2008-01-01

    Poly-ADP-ribosylation (PAR) of proteins by poly(ADP-ribose) polymerases (PARP) occurs after experimental traumatic brain injury (TBI) and modulates neurological outcome. Several promising pharmacological PARP inhibitors have been developed for use in humans, but there is currently no clinically relevant means of monitoring treatment effects. We therefore utilized an enzyme-linked immunosorbent assay (ELISA) to measure PAR-modified proteins in cerebrospinal fluid (CSF). CSF samples from 17 pediatric TBI and 15 control patients were plated overnight then incubated with polyclonal antibody against PAR. Histone-1, a PARP substrate, was incubated with active PARP, NAD, and nicked DNA, and served as the standard. Both peak and mean CSF PAR-modified protein were increased in TBI patients versus controls. Peak CSF PAR-modified protein levels occurred on day 1 and levels remained increased on day 2 after TBI. Increases in peak CSF PAR-modified protein concentrations were independently associated with age and male sex, but not initial Glasgow coma scale score, Glasgow outcome score, or mechanism of injury. The increase in PAR-modified proteins in CSF after TBI may be due to increased PARP activation, decreased PAR degradation, or both. Since PAR-modified protein concentration correlated with age and male sex, developmental and sex-dependent roles for PARP after TBI are implicated. PMID:18506195

  9. Identification, quantification, and functional aspects of skeletal muscle protein-carbonylation in vivo during acute oxidative stress.

    PubMed

    Fedorova, Maria; Kuleva, Nadezhda; Hoffmann, Ralf

    2010-05-01

    Reactive oxidative species (ROS) play important roles in cellular signaling but can also modify and often functionally inactivate other biomolecules. Thus, cells have developed effective enzymatic and nonenzymatic strategies to scavenge ROS. However, under oxidative stress, ROS production is able to overwhelm the scavenging systems, increasing the levels of functionally impaired proteins. A major class of irreversible oxidative modifications is carbonylation, which refers to reactive carbonyl-groups. In this investigation, we have studied the production and clearance rates for skeletal muscle proteins in a rat model of acute oxidative stress over a time period of 24 h using a gel-based proteomics approach. Optimized ELISA and Western blots with 10-fold improved sensitivities showed that the carbonylation level was stable at 4 nmol per mg protein 3 h following ROS induction. The carbonylation level then increased 3-fold over 6 h and then remained stable. In total, the oxidative stress changed the steady state levels of 20 proteins and resulted in the carbonylation of 38 skeletal muscle proteins. Carbonylation of these proteins followed diverse kinetics with some proteins being highly carbonylated very quickly, whereas others peaked in the 9 h sample or continued to increase up to 24 h after oxidative stress was induced. PMID:20377239

  10. Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click-SILAC approach.

    PubMed

    Somasekharan, Syam Prakash; Stoynov, Nikolay; Rotblat, Barak; Leprivier, Gabriel; Galpin, Jason D; Ahern, Christopher A; Foster, Leonard J; Sorensen, Poul H B

    2012-12-21

    Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.

  11. PTMSearchPlus: Software Tool for Automated Protein Identification and Post-Translational and Post-Translational Modification Characterization by Integrating Accurate Intact Protein Mass and Bottom-Up Mass Spectrometric Data Searches

    SciTech Connect

    Kertesz, Vilmos; Connelly, Heather M; Erickson, Brian K; Hettich, Robert {Bob} L

    2009-01-01

    PTMSearchPlus is a software tool for the automated integration of accurate intact protein mass (AIPM) and bottom-up (BU) mass spectra searches/data in order to both confidently identify the intact proteins and to characterize their post-translational modifications (PTMs). The development of PTMSearchPlus was motivated by the desire to effectively integrate high-resolution intact protein molecular masses with bottom-up peptide MS/MS data. PTMSearchPlus requires as input both intact protein and proteolytic peptide mass spectra collected from the same protein mixture, a FASTA protein database, and a selection of possible PTMs, the types and ranges of which can be specified. The output of PTMSearchPlus is a list of intact and modified proteins matching the AIPM data concomitant with their respective peptides found by the BU search. This list also contains protein and peptide sequence coverage information, scores, etc. that can be used for further evaluation or refiltering of the results. Corresponding and annotated AIPM and BU mass spectra are also displayed for visual inspection when a listed protein or a peptide is selected. These and other controls ensure that the user can manually evaluate, modify (e.g., remove obvious false positives, low quality spectra etc.), and save the results of the automated search if necessary. Driven by the exponential growth in the number of possible peptide candidates in a BU search when multiple PTMs are probed, the advantages on search speed by limiting the total number of possible PTMs on a peptide in the BU search or by performing an AIPM predicted BU search are also discussed in addition to the integration approach. The features of PTMSearchPlus are demonstrated using both a protein standard mixture and a complex protein mixture from Escherichia coli. Experimental data revealed a unique advantage of coupling AIPM and the BU data sets that is mutually beneficial for both approaches. Namely, AIPM data can confirm that no PTM peptides

  12. PTMSearchPlus: A Software Tool for Automated Protein Identification and Post-Translational Modification Characterization by Integrating Accurate Intact Protein Mass and Bottom-Up Mass Spectrometric Data Searches

    SciTech Connect

    Kertesz, Vilmos; Connelly, Heather M; Erickson, Brian K; Hettich, Robert {Bob} L

    2009-01-01

    PTMSearchPlus is a software tool for the automated integration of accurate intact protein mass (AIPM) and bottom-up (BU) mass spectra searches/data in order to both confidently identify the intact proteins and to characterize their post-translational modifications (PTMs). The development of PTMSearchPlus was motivated by the desire to effectively integrate high resolution intact protein molecular masses with bottom-up peptide MS/MS data. PTMSearchPlus requires as input both intact protein and proteolytic peptide mass spectra collected from the same protein mixture, a FASTA protein database, and a selection of possible PTMs, the types and ranges of which can be specified. The output of PTMSearchPlus is a list of intact and modified proteins matching the AIPM data concomitant with their respective peptides found by the BU search. This list also contains protein and peptide sequence coverage information, scores, etc. that can be used for further evaluation or refiltering of the results. Corresponding and annotated AIPM and BU mass spectra are also displayed for visual inspection when a listed protein or a peptide is selected. These and other controls ensure that the user can manually evaluate, modify (e.g. remove obvious false positives, low quality spectra etc.), and save the results of the automated search if necessary. Driven by the exponential growth in the number of possible peptide candidates in a BU search when multiple PTMs are probed, the advantages on search speed by limiting the total number of possible PTMs on a peptide in the BU search or by performing an AIPM predicted BU search are also discussed in addition to the integration approach. The features of PTMSearchPlus are demonstrated using both a protein standard mixture and a complex protein mixture from Escherichia coli. Experimental data revealed a unique advantage of coupling AIPM and the BU datasets that is mutually beneficial for both approaches. Namely, AIPM data can confirm that no PTM peptides

  13. A Protein-Corona-Free T(1)-T(2) Dual-Modal Contrast Agent for Accurate Imaging of Lymphatic Tumor Metastasis.

    PubMed

    Zhou, Zijian; Liu, Hanyu; Chi, Xiaoqin; Chen, Jiahe; Wang, Lirong; Sun, Chengjie; Chen, Zhong; Gao, Jinhao

    2015-12-30

    Precise nodal staging is particularly important to guide the treatments and determine the prognosis for cancer patients. However, it is still challenging to noninvasively and precisely detect in-depth tumor metastasis in lymph nodes (LNs) because of the small size and high potential of obtaining pseudopositive results. Herein, we report the rational design of a T1-T2 dual-modal MRI contrast agent for accurate imaging of tumor metastasis in LNs using gadolinium-embedded iron oxide nanoplates (GdIOP). The GdIOP were modulated with suitable size in vivo through surface functionalization by zwitterionic dopamine sulfonate (ZDS) molecules. The efficient uptake of GdIOP@ZDS nanoparticles through drainage effect because of the presence of large amount of macrophages and dendritic cells generates both T1 and T2 contrasts in LNs. In contrast, the low uptake of protein-corona-free GdIOP@ZDS nanoparticles by melanoma B16 tumor cells promises pseudocontrast imaging of potential tumor metastasis in LNs. The combination of T1 and T2 imaging modalities allows self-confirmed detection of a metastatic tumor with about 1.2 mm in the minimal dimension in LNs, which is close to the detection limit of submilimeter level of MRI scans. This study provides an efficient and noninvasive strategy to detect tumor metastasis in LNs with greatly enhanced diagnostic accuracy. PMID:26645884

  14. Quantification of horse plasma proteins altered by xylazine using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry

    PubMed Central

    MORI, Miwako; ICHIBANGASE, Tomoko; YAMASHITA, Shozo; KIJIMA-SUDA, Isao; KAWAHARA, Masahiro; IMAI, Kazuhiro

    2016-01-01

    ABSTRACT In the doping tests currently used in horse racing, prohibited substances or their metabolites are usually directly detected in urine or blood samples. However, despite their lasting pharmaceutical effects, some prohibited substances are rapidly eliminated from horse urine and blood, making them difficult to detect. Therefore, new indirect biomarkers for doping, such as plasma proteins that are increased by the prohibited substances, have recently attracted much attention. Here, a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method was adopted for horse plasma proteomics analysis, in order to identify plasma proteins whose concentrations were altered in response to xylazine in Thoroughbred horses. Xylazine, which is rapidly absorbed and eliminated and has possibility of the change in the levels of plasma proteins, was selected as a model drug. Of the ten plasma proteins identified, four proteins, including three acute phase proteins (haptoglobin, ceruloplasmin, and α-2-macroglobulin-like), were significantly increased after xylazine administration. Therefore, our present approach might be useful in identifying indirect biomarkers of drug administration. PMID:26858580

  15. Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion

    PubMed Central

    2010-01-01

    Background The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein. Results To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA). LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity. Conclusions We show that

  16. Spectroscopic characterization of protein-wrapped single-wall carbon nanotubes and quantification of their cellular uptake in multiple cell generations

    NASA Astrophysics Data System (ADS)

    Bertulli, Cristina; Beeson, Harry J.; Hasan, Tawfique; Huang, Yan Yan S.

    2013-07-01

    We study the spectral characteristics of bovine serum albumin (BSA) protein conjugated single-wall carbon nanotubes (SWNTs), and quantify their uptake by macrophages. The binding of BSA onto the SWNT surface is found to change the protein structure and to increase the doping of the nanotubes. The G-band Raman intensity follows a well-defined power law for SWNT concentrations of up to 33 μg ml-1 in aqueous solutions. Subsequently, in vitro experiments demonstrate that incubation of BSA-SWNT complexes with macrophages affects neither the cellular growth nor the cellular viability over multiple cell generations. Using wide spot Raman spectroscopy as a fast, non-destructive method for statistical quantification, we observe that macrophages effectively uptake BSA-SWNT complexes, with the average number of nanotubes internalized per cell remaining relatively constant over consecutive cell generations. The number of internalized SWNTs is found to be ˜30 × 106 SWNTs/cell for a 60 mm-2 seeding density and ˜100 × 106 SWNTs/cell for a 200 mm-2 seeding density. Our results show that BSA-functionalized SWNTs are an efficient molecular transport system with low cytotoxicity maintained over multiple cell generations.

  17. 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.

  18. Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

    PubMed

    Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

    2006-09-01

    An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

  19. Simultaneous quantification of oil and protein in cottonseed by low-field time-domain nuclear magnetic resonance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Modification of cottonseed quality traits is likely to be achieved through a combination of genetic modification, manipulation of nutrient allocation and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relation...

  20. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

    PubMed

    Hill, Jennifer J; Moreno, Maria J; Lam, Jean C Y; Haqqani, Arsalan S; Kelly, John F

    2009-02-01

    Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. PMID:19137551

  1. Spatial analysis and quantification of the thermodynamic driving forces in protein-ligand binding: binding site variability.

    PubMed

    Raman, E Prabhu; MacKerell, Alexander D

    2015-02-25

    The thermodynamic driving forces behind small molecule-protein binding are still not well-understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provide an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both nonpolar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the "hydrophobic effect". Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. It is notable to have the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  2. Chemical profiling and quantification of monacolins and citrinin in red yeast rice commercial raw materials and dietary supplements using liquid chromatography-accurate QToF mass spectrometry: Chemometrics application.

    PubMed

    Avula, Bharathi; Cohen, Pieter A; Wang, Yan-Hong; Sagi, Satyanarayanaraju; Feng, Wei; Wang, Mei; Zweigenbaum, Jerry; Shuangcheng, Ma; Khan, Ikhlas A

    2014-11-01

    Red yeast rice (RYR) is prepared by fermenting rice with various strains of the yeast Monascus spp of the Aspergillaceae family. Depending on the Monascus strains and the fermentation conditions, the products may contain monacolins, pigments and citrinin as secondary metabolites. Authentic and commercial RYR samples were analyzed using UHPLC-DAD-QToF-MS for monacolins, pigments and citrinin. A separation by UHPLC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. Accurate mass QToF spectrometry was used to distinguish isobaric monacolins. Principle component analysis (PCA), a chemometric technique was used to discriminate between authentic RYR, commercial RYR raw materials and dietary supplements. Three authentic RYR samples, 31 commercial RYR raw materials and 14 RYR dietary supplements were analyzed. Monacolin K content in 600mg of authentic RYR samples ranged from 1.2mg to 1.38mg. Amounts of monacolin K in dietary supplements labeled as containing 600mg of RYR varied more than 40-fold from 0.03mg to 2.18mg. Monacolin K content of dietary supplements labeled as containing 1200mg RYR varied more than 20-fold from 0.22mg to 5.23mg. In addition to large variations in quantity of monacolin K found in dietary supplements, RYR dietary supplements contained ratios of monacolins that differed significantly from authentic samples. The results indicated that RYR commercial products are of variable quality and the analytical method is suitable for quality control testing of a variety of RYR products.

  3. Novel and cost-effective 6-plex isobaric tagging reagent, DiART, is effective for identification and relative quantification of complex protein mixtures using PQD fragmentation.

    PubMed

    Ramsubramaniam, Nikhil; Tao, Feng; Li, Shuwei; Marten, Mark R

    2013-09-01

    Deuterium isobaric Amine Reactive Tag (DiART) reagents facilitate relative quantification during proteomic analysis in a functionally similar manner to commercially available isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) reagents. In contrast to iTRAQ and TMT, DiART reagents incorporate deuterium isotopes which significantly reduce the number of required synthesis steps and hence have potential to significantly reduce reagent production cost. We examined the capability of DiART for performing quantitative proteomic experiments using a linear ion-trap mass spectrometer with Pulsed Q Dissociation (PQD) fragmentation. Using a synthetic peptide tagged with DiART reagent, we observed a precise mass shift of 144.79 Da on the triply charged precursor ion, which shows complete derivatization of the N-terminus and ε-amino group of lysine. A DiART tagged tryptic digest of bovine serum albumin showed a sequence coverage of 57.99% which was very comparable to that showed by iTRAQ, 54.77%. Furthermore, a ten protein mixture tagged with DiART reagents and mixed in 1:1:1:1:1:1 exhibited < 15% error, whereas a linear trend (slope of 1.085) was observed when tagged proteins were mixed in the ratio 2:1:2:4:10:14 and plotted against theoretical ratios. Finally, when complex cell-wall protein mixtures from the model fungus A. nidulans were tagged with DiART reagents and mixed in different ratios, they exhibited similar trends. We conclude that DiART reagents are capable of performing quantitative proteomic experiments using PQD on a linear ion trap mass spectrometer.

  4. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    NASA Astrophysics Data System (ADS)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  5. Development of visible diode-laser/fiber-optic colorimetric system for the simple and inexpensive quantification of a protein

    NASA Astrophysics Data System (ADS)

    Kim, SungHo; Noh, Young S.; Byun, Gill S.; Jang, Sung-Keun

    1998-04-01

    A simple and inexpensive quantitative detection of protein in one droplet solution was achieved by a portable diode-laser- based-optical-fiber absorption spectrometry. A system consisted of a visible diode laser, a photodiode and a pair of optical fibers. The conventional Lowry method was used to measure the blue-colored protein solutions. In this study, one droplet of sample, which filled up the void between the end surfaces of two optical fibers, served as a sample cell. The volume of a droplet was 50 (mu) L, which is 1/100 of the cuvette of UV/VIS spectrometer cell used in Lowry method. Although the required sample volume decreased up to 100 times, the detection range was comparable to that of conventional Lowry method in the range of g/L - mg/L.

  6. Quantification of major royal jelly protein 1 in fresh royal jelly by indirect enzyme-linked immunosorbent assay.

    PubMed

    Yamaguchi, Kikuji; He, Shaoyu; Li, Zhengyue; Murata, Kiyoshi; Hitomi, Nobuyuki; Mozumi, Manaho; Ariga, Risa; Enomoto, Toshiki

    2013-01-01

    Rabbit polyclonal antibody produced by a major royal jelly protein 1 (MRJP1) specific peptide reacted only with a MRJP1. Indirect ELISA with the antibody revealed a MRJP1 level of 4.12-4.67 g/100 g in different company's royal jelly, which almost agreed with that of a hexametric form of MRJP1 (apisin) measured by high performance liquid chromatography. These results suggest that MRJP1 exists mainly as apisin in royal jelly.

  7. Expression patterns of prion protein gene in differential genotypes sheep: quantification using molecular beacon real-time RT-PCR.

    PubMed

    Wang, Chuan; Wu, Run; Li, Fa-Di; Liu, Lei; Zhang, Xiao-Li; Zhao, Chun-Lin; Diao, Xiao-Long; Guan, Hong-Wei

    2011-06-01

    Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its role in organisms and revealing mechanism of susceptibility and resistance to scrapie. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail in digestive tract which is important during scrapie spread through oral route. Herein, we report on measurement of sheep PrP mRNA using absolute quantitative real-time RT-PCR. Total RNA was isolated from five different regions of the central nervous system (CNS), four regions of lymphoid system, eleven regions of digestive tract, and two reproductive organ tissues of eight sheep of two different genotypes (ARR/ARQ and ARH/ARQ) and PrP mRNA was quantified by real-time RT-PCR using molecular beacon. The results showed that highest levels of PrP mRNA were expressed in thalamus and cerebrum (P < 0.01) of CNS examined, followed by cerebellum, spinal cord, and brain stem. In peripheral organs examined, lymph tissue showed moderate level of PrP expression similar to that in digestive tract and reproduction organs. PrP expression levels in the same tissue of different genotype sheep had significant variation. Our study provided the first detail, tissue-specific and genotype-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.

  8. Quantification of oxidative stress phenotypes based on high-throughput growth profiling of protein kinase and phosphatase knockouts.

    PubMed

    Altıntaş, Ali; Martini, Jacopo; Mortensen, Uffe H; Workman, Christopher T

    2016-02-01

    Cellular responses to oxidative stress are important for restoring redox balance and ensuring cell survival. Genetic defects in response factors can lead to impaired response to oxidative damage and contribute to disease and aging. In single cell organisms, such as yeasts, the integrity of the oxidative stress response can be observed through its influences on growth characteristics. In this study, we investigated the time-dependent batch growth effects as a function of oxidative stress levels in protein kinase and phosphatase deletion backgrounds of Saccharomyces cerevisiae. In total, 41 different protein kinases and phosphatase mutants were selected for their known activities in oxidative stress or other stress response pathways and were investigated for their dosage-dependent response to hydrogen peroxide. Detailed growth profiles were analyzed after the induction of stress for growth rate, lag time duration and growth efficiency, and by a novel method to identify stress-induced diauxic shift delay. This approach extracts more phenotypic information than traditional plate-based methods due to the assessment of time dynamics in the time scale of minutes. With this approach, we were able to identify surprisingly diverse sensitivity and resistance patterns as a function of gene knockout.

  9. Data-independent liquid chromatography/mass spectrometry (LC/MS(E)) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase.

    PubMed

    Blackburn, Kevin; Cheng, Fang-Yi; Williamson, John D; Goshe, Michael B

    2010-04-15

    Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis.

  10. In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

    PubMed Central

    Aubele, M; Spears, M; Ludyga, N; Braselmann, H; Feuchtinger, A; Taylor, K J; Lindner, K; Auer, G; Stering, K; Höfler, H; Schmitt, M; Bartlett, J M S

    2010-01-01

    Background: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. Method and results: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). Conclusion: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance. PMID:20700126

  11. An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum.

    PubMed

    Chhatre, Sunil; Francis, Richard; Bracewell, Daniel G; Titchener-Hooker, Nigel J

    2010-11-15

    The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.

  12. Analysis of tandem mass spectra by FTMS for improved large-scale proteomics with superior protein quantification

    PubMed Central

    McAlister, Graeme C.; Phanstiel, Doug; Wenger, Craig D.; Lee, M. Violet; Coon, Joshua J.

    2009-01-01

    Using a newly developed dual-cell quadrupole linear ion trap-orbitrap hybrid mass spectrometer (dcQLT-orbitrap), we demonstrate the utility of collecting high-resolution tandem mass spectral data for large-scale shotgun proteomics. Multiple nanoLC-MS/MS experiments on both an older generation quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) and the dcQLT-orbitrap, using both resonant-excitation CAD and beam-type CAD (HCD) were performed. Resulting from various technological advances (e.g., a stacked ring ion guide AP inlet, a dual cell QLT, etc.), the dcQLT-orbitrap exhibited increased duty cycle (~1.5–2×) and sensitivity for both CAD (ion trap detection) and HCD (orbitrap detection) methods. As compared to the older system, the dcQLT-orbitrap produced significantly more unique peptide identification for both methods (~30% improvement for CAD and ~115% improvement for HCD). The sizeable improvement of the HCD method on the dcQLT-orbitrap system, outperforms the current standard method of CAD with ion trap detection for large-scale analysis. Finally, we demonstrate that the increased HCD performance translates to a direct and substantial improvement in protein quantitation precision using isobaric tags. PMID:19938823

  13. Biomarker assay translation from discovery to clinical studies in cancer drug development: quantification of emerging protein biomarkers.

    PubMed

    Lee, Jean W; Figeys, Daniel; Vasilescu, Julian

    2007-01-01

    Many candidate biomarkers emerging from genomics and proteomics research have the potential to serve as predictive indexes for guiding the development of safer and more efficacious drugs. Research and development of biomarker discovery, selection, and clinical qualification, however, is still a relatively new field for the pharmaceutical industry. Advances in technology provide a plethora of analytical tools to discover and analyze mechanism-and-disease-specific biomarkers for drug development. In the discovery phase, differential proteomic analysis using mass spectrometry enables the identification of candidate biomarkers that are associated with a specific mechanism relevant to disease progression and affected by drug treatment. Reliable bioanalytical methods are then developed and implemented to select promising biomarkers for further studies in animals and humans. Quantitative analytical methods capable of generating reliable data constitute a solid basis for statistical assessment of the predictive utility of biomarkers. Biomarker method validation is diverse and for purposes that are very different from those of drug bioanalysis or diagnostic use. Besides being flexible, it should sufficiently demonstrate the method's ability to meet the study intent and the attendant regulatory requirements. Several papers have been published outlining specific requirements for successful biomarker method development and validation using a "Fit-for-Purpose" approach. Many of the challenges faced during biomarker discovery as well as during technology and process translation are discussed in this chapter, including preanalytical planning, assay development, and preclinical and clinical validation. Specific references to protein biomarkers for cancer drug development are also discussed.

  14. Quantification of Plasmodium falciparum histidine-rich protein-2 in cerebrospinal spinal fluid from cerebral malaria patients.

    PubMed

    Mikita, Kei; Thakur, Kiran; Anstey, Nicholas M; Piera, Kim A; Pardo, Carlos A; Weinberg, J Brice; Mukemba, Jackson; Florence, Salvatore; Mwaikambo, Esther D; Granger, Donald L; Sullivan, David J

    2014-09-01

    A cerebrospinal fluid (CSF) biomarker for cerebral malaria (CM) has not been validated. We examined the detection, semiquantification, and clinical use of the Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) as a parasite antigen biomarker for CM. The PfHRP-2 was detected in archival CSF samples from CM patients from Tanzania both by a newly developed sensitive and specific immuno-polymerase chain reaction (72 of 73) and by rapid diagnostic tests (62 of 73). The geometric mean PfHRP-2 CSF concentration was 8.76 ng/mL with no differences in those who survived (9.2 ng/mL), those who died (11.1 ng/mL), and those with neurologic sequelae (10.8 ng/mL). All aparasitemic endemic and nonendemic control samples had undetectable CSF PfHRP-2. In a separate group of 11 matched plasma and CSF cerebral malaria patient samples, the ratio of plasma to CSF PfHRP-2 was 175. The CSF PfHRP-2 reflects elevated plasma PfHRP-2 rather than elevated CM-specific CSF ratios, falling short of a validated biomarker.

  15. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry.

    PubMed

    Hartmann, Erica M; Colquhoun, David R; Schwab, Kellogg J; Halden, Rolf U

    2015-04-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences.

  16. Prediction of recurrence by quantification of p185neu protein in non-small-cell lung cancer tissue.

    PubMed Central

    Diez, M.; Pollán, M.; Maestro, M.; Torres, A.; Ortega, D.; Gómez, A.; Sánchez, A.; Hernando, F.; Balibrea, J. L.

    1997-01-01

    The concentration of c-erbB-2 oncogene-encoded protein (p185neu) in fresh tumour samples obtained at the time of surgery from 94 non-small-cell lung cancer patients (NSCLC) was determined by an enzyme immunoassay. The relative prognostic importance was estimated, and the influence of other predictors was assessed by means of a Cox's proportional regression model. Median concentration of p185 in tumour tissues was 206 U mg(-1) (range 21-1050 U mg(-1)). p185 level did not differ significantly among subgroups defined by TNM classification, histological type, sex and age. Categorization of patients by p185 level, with 206 U mg(-1) and 343 U mg(-1) taken as cut-off values (corresponding to the 50th and 80th percentiles of the frequency distribution), showed that the recurrence rate, cumulative disease-free likelihood at the 36-month follow-up and median time from surgery to the diagnosis of recurrence worsened progressively as the level of p185 increased. Multivariate analysis confirmed the independent prognostic value of p185 level. Risk of recurrence increased by 1.304 for every increase of 100 units in p185 concentration (95% CI 1.141-1.490) (P<0.001). These findings encourage the inclusion of p185 concentration assay in a future predictive multifactorial prognostic index in NSCLC. PMID:9043025

  17. Absolute Quantification of Norovirus Capsid Protein in Food, Water, and Soil Using Synthetic Peptides with Electrospray and MALDI Mass Spectrometry

    PubMed Central

    Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

    2015-01-01

    Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrixassisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences. PMID:25603302

  18. A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management

    PubMed Central

    Jagarlamudi, Kiran Kumar; Moreau, Laura; Westberg, Sara; Rönnberg, Henrik; Eriksson, Staffan

    2015-01-01

    Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (rs = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology. PMID:26366881

  19. A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management.

    PubMed

    Jagarlamudi, Kiran Kumar; Moreau, Laura; Westberg, Sara; Rönnberg, Henrik; Eriksson, Staffan

    2015-01-01

    Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (rs = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology.

  20. UHPLC-MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients.

    PubMed

    De Nicolò, Amedeo; Avataneo, Valeria; Rabbia, Franco; Bonifacio, Gabriele; Cusato, Jessica; Tomasello, Cristina; Perlo, Elisa; Mulatero, Paolo; Veglio, Franco; Di Perri, Giovanni; D'Avolio, Antonio

    2016-09-10

    Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity(®) UPLC HSS T3 1.8μm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy. PMID:27497654

  1. Quantification of particle-conjugated or -encapsulated peptides on interfering reagent backgrounds

    PubMed Central

    Yap, Woon Teck; Song, W. Kelsey; Chauhan, Niharika; Scalise, P. Nina; Agarwal, Radhika; Shea, Lonnie D.

    2014-01-01

    Particle-based technologies are increasingly used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide while dimethyl sulfoxide is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on the bicinchoninic acid, Coomassie Plus and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde assay was a highly sensitive and accurate substitute for radioactivity, suitable for measuring multiple particle-bound or -encapsulated peptides or proteins in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or poly(lactide-co-glycolide) in dimethyl sulfoxide that interfere with the more commonly-used bicinchoninic acid and Coomassie Plus assays. Our strategy enables the accurate quantification of peptides or proteins loaded onto or into particles – an essential component of designing particle-based platforms for diagnostics and therapeutics. PMID:25005692

  2. Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

    PubMed

    Liu, Jason Yingjie

    2014-11-01

    The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA.

  3. Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.

    PubMed

    Kao, Athit; Chiu, Chi-li; Vellucci, Danielle; Yang, Yingying; Patel, Vishal R; Guan, Shenheng; Randall, Arlo; Baldi, Pierre; Rychnovsky, Scott D; Huang, Lan

    2011-01-01

    Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

  4. A 3D Time-Shared NOESY Experiment Designed to Provide Optimal Resolution for Accurate Assignment of NMR Distance Restraints in Large Proteins

    PubMed Central

    Mishra, Subrata H; Harden, Bradley J

    2014-01-01

    Structure determination of proteins by solution NMR has become an established method, but challenges increase steeply with the size of proteins. Notably spectral crowding and signal overlap impair the analysis of cross-peaks in NOESY spectra that provide distance restraints for structural models. An optimal spectral resolution can alleviate overlap but requires prohibitively long experimental time with existing methods. Here we present a time-shared 3D experiment optimized for large proteins that provides 15N and 13C dispersed NOESY spectra in a single measurement. NOESY correlations appear in the detected dimension and hence benefit from the highest resolution achievable of all dimensions without increase in experimental time. By design, this experiment is inherently optimal for non-uniform sampling acquisition when compared to current alternatives. Thus, 15N and 13C dispersed NOESY spectra with ultra-high resolution in all dimensions were acquired in parallel within about 4 days instead of 80 days for a 52 kDa monomeric protein at a concentration of 350 μM. PMID:25381567

  5. Asymmetric Flow-Field Flow Fractionation Hyphenated ICP-MS as an Alternative to Cloud Point Extraction for Quantification of Silver Nanoparticles and Silver Speciation: Application for Nanoparticles with a Protein Corona.

    PubMed

    Mudalige, Thilak K; Qu, Haiou; Linder, Sean W

    2015-07-21

    Production and application of nanoparticles in consumer products is at an all-time high due to the emerging field of nanotechnology. Direct detection and quantification of trace levels of nanoparticles within consumer products is very challenging and problematic. Although multiple methodologies are available for this purpose, each method has its own set of limitations. Herein, we developed an analytical platform consisting of asymmetric flow-field flow fractionation (AF4) coupled with inductively coupled plasma mass spectroscopy (ICP-MS) for the speciation and quantification of silver ions and silver nanoparticles at the ng/kg level (ppt). AF4 is utilized to concentrate the nanoparticles, and ICP-MS acts as the detector. The protein corona that forms upon exposure of nanoparticles to bovine serum albumin was utilized as a nanoparticle stabilization and AF4 recovery enhancement mechanism. Speciation of silver ions and nanoparticles was achieved with the assistance of penicillamine as a complexation ligand. The effect of nanoparticle size, surface coating, and ionization state toward the detection and quantification of the developed methodology was evaluated. The detection limit was found to be 4 ng/kg with the application of a 5 mL sample loop. Further application of this developed methodology on environmentally relevant samples was demonstrated by the analysis of Arkansas River water spiked with silver nanoparticles and nanoparticle spiked into humic acid solution (50 mg/L) at an environmentally relevant level.

  6. Separation and quantification of microalgal carbohydrates.

    PubMed

    Templeton, David W; Quinn, Matthew; Van Wychen, Stefanie; Hyman, Deborah; Laurens, Lieve M L

    2012-12-28

    Structural carbohydrates can constitute a large fraction of the dry weight of algal biomass and thus accurate identification and quantification is important for summative mass closure. Two limitations to the accurate characterization of microalgal carbohydrates are the lack of a robust analytical procedure to hydrolyze polymeric carbohydrates to their respective monomers and the subsequent identification and quantification of those monosaccharides. We address the second limitation, chromatographic separation of monosaccharides, here by identifying optimum conditions for the resolution of a synthetic mixture of 13 microalgae-specific monosaccharides, comprised of 8 neutral, 2 amino sugars, 2 uronic acids and 1 alditol (myo-inositol as an internal standard). The synthetic 13-carbohydrate mix showed incomplete resolution across 11 traditional high performance liquid chromatography (HPLC) methods, but showed improved resolution and accurate quantification using anion exchange chromatography (HPAEC) as well as alditol acetate derivatization followed by gas chromatography (for the neutral- and amino-sugars only). We demonstrate the application of monosaccharide quantification using optimized chromatography conditions after sulfuric acid analytical hydrolysis for three model algae strains and compare the quantification and complexity of monosaccharides in analytical hydrolysates relative to a typical terrestrial feedstock, sugarcane bagasse.

  7. Functionalized quantum dots with dopamine dithiocarbamate as the matrix for the quantification of efavirenz in human plasma and as affinity probes for rapid identification of microwave tryptic digested proteins in MALDI-TOF-MS.

    PubMed

    Kailasa, Suresh Kumar; Wu, Hui-Fen

    2012-06-01

    Functionalized quantum dots with dopamine dithiocarbamate (QDs-DDTC) were utilized for the first time as an efficient material for the quantification of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digest proteins (cytochrome c, lysozyme and BSA) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesized QDs-DDTC was characterized by using spectroscopic (UV-visible, FT-IR and (1)H NMR) and microscopic (SEM and TEM) techniques. Functionalized QDs-DDTC exhibited a high desorption/ionization efficiency for the rapid quantification of small molecules (efavirenz, tobramycin and aspartame) at low-mass region. QDs-DDTC has well ability to trap target species, and capable to transfer laser energy for efficient desorption/ionization of analytes with background-free detection. The use of QDs-DDTC as a matrix provided good linearity for the quantification of small molecules (R(2)=~0.9983), with good reproducibility (RSD<10%), in the analysis of efavirenz in the plasma of HIV infected patients by the standard addition method. We also demonstrated that the use of functionalized QDs-DDTC as affinity probes for the rapid identification of microwave tryptic digested proteins (cytochrome c, lysozyme and BSA) by MALDI-TOF-MS. QDs-DDTC-based MALDI-TOF-MS approach provides simplicity, rapidity, accuracy, and precision for the determination of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digested proteins. This new material presents a marked advance in the development of matrix-free mass spectrometric methods for the rapid and precise quantitative determination of a variety of molecules. This article is part of a Special Issue entitled: Proteomics: The clinical link.

  8. A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection.

    PubMed

    Otsuka, Nao; Gotoh, Kensei; Nishimura, Naoko; Ozaki, Takao; Nakamura, Yukitsugu; Haga, Kiyohito; Yamazaki, Makoto; Gondaira, Fumio; Okada, Kenji; Miyaji, Yusuke; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Arakawa, Yoshichika; Kamachi, Kazunari

    2016-05-01

    An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

  9. Accurate theoretical prediction of vibrational frequencies in an inhomogeneous dynamic environment: A case study of a glutamate molecule in water solution and in a protein-bound form

    NASA Astrophysics Data System (ADS)

    Speranskiy, Kirill; Kurnikova, Maria

    2004-07-01

    We propose a hierarchical approach to model vibrational frequencies of a ligand in a strongly fluctuating inhomogeneous environment such as a liquid solution or when bound to a macromolecule, e.g., a protein. Vibrational frequencies typically measured experimentally are ensemble averaged quantities which result (in part) from the influence of the strongly fluctuating solvent. Solvent fluctuations can be sampled effectively by a classical molecular simulation, which in our model serves as the first, low level of the hierarchy. At the second high level of the hierarchy a small subset of system coordinates is used to construct a patch of the potential surface (ab initio) relevant to the vibration in question. This subset of coordinates is under the influence of an instantaneous external force exerted by the environment. The force is calculated at the lower level of the hierarchy. The proposed methodology is applied to model vibrational frequencies of a glutamate in water and when bound to the Glutamate receptor protein and its mutant. Our results are in close agreement with the experimental values and frequency shifts measured by the Jayaraman group by the Fourier transform infrared spectroscopy [Q. Cheng et al., Biochem. 41, 1602 (2002)]. Our methodology proved useful in successfully reproducing vibrational frequencies of a ligand in such a soft, flexible, and strongly inhomogeneous protein as the Glutamate receptor.

  10. Quantification of Tryptic Peptides in Quadrupole Ion Trap Using High-Mass Signals Derived from Isotope-Coded N-Acetyl Dipeptide Tags

    NASA Astrophysics Data System (ADS)

    Seo, Jongcheol; Yoon, Hye-Joo; Shin, Seung Koo

    2011-09-01

    Isotope-labeled N-acetyl dipeptides (Ac-Xxx-Ala) are coupled to the primary amines of tryptic peptides and then analyzed by tandem mass spectrometry. Amide bond cleavage between Xxx and Ala provides both low- and high-mass isotope-coded signals for quantification of peptides. Especially, facile cleavage at the modified lysine side chain yields very strong high-mass quantitation signals in a noise-free region. Tagging tryptic peptides with isobaric N-acetyl dipeptides is a viable strategy for accurate quantification of proteins, which can be used with most quadrupole ion trap mass spectrometers carrying the 1/3 mass cut-off problem.

  11. The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement.

    PubMed

    May, Sophie F; Peacock, Lori; Almeida Costa, Cristina I C; Gibson, Wendy C; Tetley, Laurence; Robinson, Derrick R; Hammarton, Tansy C

    2012-04-01

    AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.

  12. Accurate measurements of dynamics and reproducibility in small genetic networks

    PubMed Central

    Dubuis, Julien O; Samanta, Reba; Gregor, Thomas

    2013-01-01

    Quantification of gene expression has become a central tool for understanding genetic networks. In many systems, the only viable way to measure protein levels is by immunofluorescence, which is notorious for its limited accuracy. Using the early Drosophila embryo as an example, we show that careful identification and control of experimental error allows for highly accurate gene expression measurements. We generated antibodies in different host species, allowing for simultaneous staining of four Drosophila gap genes in individual embryos. Careful error analysis of hundreds of expression profiles reveals that less than ∼20% of the observed embryo-to-embryo fluctuations stem from experimental error. These measurements make it possible to extract not only very accurate mean gene expression profiles but also their naturally occurring fluctuations of biological origin and corresponding cross-correlations. We use this analysis to extract gap gene profile dynamics with ∼1 min accuracy. The combination of these new measurements and analysis techniques reveals a twofold increase in profile reproducibility owing to a collective network dynamics that relays positional accuracy from the maternal gradients to the pair-rule genes. PMID:23340845

  13. First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

    PubMed

    Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

    2007-02-21

    An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

  14. First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

    PubMed

    Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

    2007-02-21

    An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis. PMID:17300145

  15. Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line.

    PubMed

    Cutillas, Pedro R; Geering, Barbara; Waterfield, Mike D; Vanhaesebroeck, Bart

    2005-08-01

    simple LC-MS method for quantification of gel-separated proteins and their phosphorylation sites and for quantitative profiling of biological systems.

  16. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

    PubMed

    Nakamura, Kenji; Hirayama-Kurogi, Mio; Ito, Shingo; Kuno, Takuya; Yoneyama, Toshihiro; Obuchi, Wataru; Terasaki, Tetsuya; Ohtsuki, Sumio

    2016-08-01

    The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

  17. Continuous nanoflow-scanning electrochemical microscopy: voltammetric characterization and application for accurate and reproducible imaging of enzyme-labeled protein microarrays.

    PubMed

    Kai, Tianhan; Chen, Shu; Monterroso, Estuardo; Zhou, Feimeng

    2015-04-21

    The coupling of scanning electrochemical microscopy (SECM) to a continuous nanoflow (CNF) system is accomplished with the use of a microconcentric ring electrode/injector probe. The gold microring electrode encapsulated by a glass sheath is robust and can be beveled and polished. The CNF system, comprising a precision gas displacement pump and a rotary valve, is capable of delivering solution to the center of the SECM probe in the range of 1-150 nL/min. Major advantages of the CNF-SECM imaging mode over the conventional SECM generation/collection (G/C) mode include higher imaging resolution, immunity from interferences by species in the bulk solution or at other sites of the substrate, elimination of the feedback current that could interfere with the G/C data interpretation, and versatility of initiating surface reactions/processes via introducing different reactants into the flowing stream. Parameters such as flow rates, probe/substrate separations, and collection efficiencies are examined and optimized. Higher resolution, reproducibility, and accuracy are demonstrated through the application of CNF-SECM to horseradish peroxidase (HRP)-amplified imaging of protein microarrays. By flowing H2O2 and ferrocenemethanol through the injector and detecting the surface-generated ferriceniummethanol, human IgG spots covered with HPR-labeled antihuman IgG can be detected in the range of 13 nM-1.333 μM with a detection limit of 3.0 nM. In addition, consistent images of microarray spots for selective and high-density detection of analytes can be attained. PMID:25831146

  18. Critical review of current and emerging quantification methods for the development of influenza vaccine candidates.

    PubMed

    Manceur, Aziza P; Kamen, Amine A

    2015-11-01

    Significant improvements in production and purification have been achieved since the first approved influenza vaccines were administered 75 years ago. Global surveillance and fast response have limited the impact of the last pandemic in 2009. In case of another pandemic, vaccines can be generated within three weeks with certain platforms. However, our Achilles heel is at the quantification level. Production of reagents for the quantification of new vaccines using the SRID, the main method formally approved by regulatory bodies, requires two to three months. The impact of such delays can be tragic for vulnerable populations. Therefore, efforts have been directed toward developing alternative quantification methods, which are sensitive, accurate, easy to implement and independent of the availability of specific reagents. The use of newly-developed antibodies against a conserved region of hemagglutinin (HA), a surface protein of influenza, holds great promises as they are able to recognize multiple subtypes of influenza; these new antibodies could be used in immunoassays such as ELISA and slot-blot analysis. HA concentration can also be determined using reversed-phase high performance liquid chromatography (RP-HPLC), which obviates the need for antibodies but still requires a reference standard. The number of viral particles can be evaluated using ion-exchange HPLC and techniques based on flow cytometry principles, but non-viral vesicles have to be taken into account with cellular production platforms. As new production systems are optimized, new quantification methods that are adapted to the type of vaccine produced are required. The nature of these new-generation vaccines might dictate which quantification method to use. In all cases, an alternative method will have to be validated against the current SRID assay. A consensus among the scientific community would have to be reached so that the adoption of new quantification methods would be harmonized between

  19. Accurate measurements of {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    SciTech Connect

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Nielsen, Niels Chr.; Khaneja, Navin

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

  20. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

    PubMed

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-12-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.

  1. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

    PubMed

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-12-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

  2. Quantification of Translocator Protein (18 kDa) in the Human Brain with PET and a Novel Radioligand, 18F-PBR06

    PubMed Central

    Fujimura, Yota; Zoghbi, Sami S.; Simèon, Fabrice G.; Taku, Andrew; Pike, Victor W.; Innis, Robert B.; Fujita, Masahiro

    2009-01-01

    Translocator protein (TSPO) (18 kDa), formerly called the peripheral benzodiazepine receptor, is upregulated on activated microglia and macrophages and is, thus, a biomarker of inflammation. We previously reported that an 11C-labeled aryloxyanilide (half-life, 20 min) was able to quantify TSPOs in the healthy human brain. Because many PET centers would benefit from a longer-lived 18F-labeled radioligand (half-life, 110 min), the objective of this study was to evaluate the ability of a closely related aryloxyanilide (18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline [18F-PBR06]) to quantify TSPOs in the healthy human brain. Methods A total of 9 human subjects were injected with 18F-PBR06 (~185 MBq) and scanned for 5 h, with rest periods outside the camera. The concentrations of 18F-PBR06, separated from radiometabolites, were measured in arterial plasma. Results Modeling of regional brain and plasma data showed that a 2-tissue-compartment model was superior to a 1-tissue-compartment model. Even if data for all time points were used for the fitting, concentrations of brain activity measured with PET were consistently greater than the modeled values at late (280–300 min) but not at early time points. The greater values may have been caused by the slow accumulation of radiometabolites in the brain. To determine an adequate time for more accurate measurement of distribution volume (VT), which is the summation of receptor binding and nondisplaceable activity, we investigated which scan duration would be associated with maximal or near-maximal identifiability. We found that a scan of 120 min provided the best identifiability of VT (~2%). The images showed no significant defluorination. Conclusion 18F-PBR06 can quantify TSPOs in the healthy human brain using 120 min of image acquisition and concurrent measurements of radioligand in plasma. Although brain activity is likely contaminated with radiometabolites, the percentage contamination is thought to be small

  3. Quantification of 4-hydroxy-2-nonenal-protein adducts in the in vivo gastric digesta of mini-pigs using a GC-MS/MS method with accuracy profile validation.

    PubMed

    Delosière, Mylène; Santé-Lhoutellier, Véronique; Chantelauze, Céline; Durand, Denys; Thomas, Agnès; Joly, Charlotte; Pujos-Guillot, Estelle; Rémond, Didier; Comte, Blandine; Gladine, Cécile; Guy, Alexandre; Durand, Thierry; Laurentie, Michel; Dufour, Claire

    2016-08-10

    Hydroxyalkenals are lipid oxidation end-products resulting from the oxidation of polyunsaturated fatty acids (PUFA). This study aimed at quantifying the production of 4-hydroxy-2-nonenal-protein adducts (HNE-P) via Michael addition from n-6 PUFA oxidation in the gastric digesta of mini-pigs after the consumption of meat-based meals with different plant antioxidant contents. Using the accuracy profile procedure, we validated an extraction protocol for the quantification of HNE-P by GC-MS/MS in gastric contents. The formation of HNE-P in the gastric compartment was observed for the first time, with concentrations ranging from less than 0.52 to 1.33 nmol HNE-P per 500 mg digesta. Nevertheless, most gastric HNE-P levels were below the limit of quantification of 0.52 nmol HNE-P per 500 mg digesta. In this animal study, the protective effect of plant antioxidant sources on HNE-P formation was not evidenced contrasting with the results using TBARS as markers. PMID:27418316

  4. Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA).

    PubMed

    Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton; Karzai, A Wali

    2013-10-18

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.

  5. Multidimensional Separation Using HILIC and SCX Pre-fractionation for RP LC-MS/MS Platform with Automated Exclusion List-based MS Data Acquisition with Increased Protein Quantification

    PubMed Central

    Zhou, Yu; Meng, Zhen; Edman-Woolcott, Maria; Hamm-Alvarez, Sarah F; Zandi, Ebrahim

    2015-01-01

    Liquid chromatography–mass spectrometry (LC-MS) based proteomics is one of the most widely used analytical platforms for global protein discovery and quantification. One of the challenges is the difficulty of identifying low abundance biomarker proteins from limited biological samples. Extensive fractionation could expand proteomics dynamic range, however, at the cost of high sample and time consumption. Extensive fractionation would increase the sample need and the labeling cost. Also quantitative proteomics depending on high resolution MS have the limitation of spectral acquisition speed. Those practical problems hinder the in-depth quantitative proteomics analysis such as tandem mass tag (TMT) experiments. We found the joint use of hydrophilic interaction liquid chromatography (HILIC) and strong cation exchange Chromatography (SCX) prefractionation at medium level could improve MS/MS efficiency, increase proteome coverage, shorten analysis time and save valuable samples. In addition, we scripted a program, Exclusion List Convertor (ELC), which automates and streamlines data acquisition workflow using the precursor ion exclusion (PIE) method. PIE reduces redundancy of high abundance MS/MS analyses by running replicates of the sample. The precursor ions detected in the initial run(s) are excluded for MS/MS in the subsequent run. We compared PIE methods with standard data dependent acquisition (DDA) methods running replicates without PIE for their effectiveness in quantifying TMT-tagged peptides and proteins in mouse tears. We quantified a total of 845 proteins and 1401 peptides using the PIE workflow, while the DDA method only resulted in 347 proteins and 731 peptides. This represents a 144% increase of protein identifications as a result of PIE analysis. PMID:26807013

  6. Quantitative peptide and protein profiling by mass spectrometry.

    PubMed

    Schmidt, Alexander; Bisle, Birgit; Kislinger, Thomas

    2009-01-01

    Proteomics may be defined as the systematic analysis of proteins expressed in a given organism (Electrophoresis 16:1090-1094, 1995). Important technical innovations in mass spectrometry (MS), protein identification methods, and database annotation, over the past decade, now make it possible to routinely identify thousands of proteins in complex biological samples (Nature 422:198-207, 2003). However, to gain new insights regarding fundamental biological questions, accurate protein quantification is also required. In this chapter, we present methods for the biochemical separation of different cellular compartments, two-dimensional chromatographic separation of the constituent peptide populations, and the recently published Spectral Counting Strategy, a label-free MS-based protein quantification technology (Cell 125:173-186, 2006; Anal Chem 76:4193-4201, 2004; Mol Cell Proteomics 4:1487-1502, 2005; Cell 125:1003-1013, 2006; Methods 40:135-142, 2006; Anal Chem 77:6218-6224, 2005; J Proteome Res 5:2339-2347, 2006). Additionally, highly accurate protein quantification based on isotope dilution, describing the isotope coded protein label (ICPL) -- method shall be explained in detail (Mol Cell Proteomics 5:1543-1558, 2006; Proteomics 5:4-15, 2005).

  7. Quantification of soy protein using the isotope method (δ(13)C and δ(15)N) for commercial brands of beef hamburger.

    PubMed

    Ducatti, Rhani; de Almeida Nogueira Pinto, José Paes; Sartori, Maria Márcia Pereira; Ducatti, Carlos

    2016-12-01

    Hamburgers (beef patties) may be adulterated through the overuse of protein extenders. Among vegetables, soy protein is the best substitute for animal protein. These ingredients help to reduce the cost of producing a final product, and they maximize profits for fraudulent industries. Moreover, the ingestion of soy or other non-meat proteins by allergic individuals may present a health risk. In addition, monitoring by supervisory bodies is hampered by a lack of appropriate analytical methodologies. Within this context, the aim of this study was to determine and quantify the levels of added soy protein by determination of (15)N and (13)C stable isotopes. A total of 100 beef hamburger samples from 10 commercial brands were analyzed. Only three samples of the G brand were within the standards set the Brazilian legislation. The remaining 97 samples from 10 commercial brands contained >4% soy protein; therefore, they are adulterated and not in compliance with the current legislation. PMID:27501234

  8. Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

    PubMed

    George, Iniga S; Fennell, Anne Y; Haynes, Paul A

    2015-09-01

    Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399).

  9. Accurate monotone cubic interpolation

    NASA Technical Reports Server (NTRS)

    Huynh, Hung T.

    1991-01-01

    Monotone piecewise cubic interpolants are simple and effective. They are generally third-order accurate, except near strict local extrema where accuracy degenerates to second-order due to the monotonicity constraint. Algorithms for piecewise cubic interpolants, which preserve monotonicity as well as uniform third and fourth-order accuracy are presented. The gain of accuracy is obtained by relaxing the monotonicity constraint in a geometric framework in which the median function plays a crucial role.

  10. Accurate Finite Difference Algorithms

    NASA Technical Reports Server (NTRS)

    Goodrich, John W.

    1996-01-01

    Two families of finite difference algorithms for computational aeroacoustics are presented and compared. All of the algorithms are single step explicit methods, they have the same order of accuracy in both space and time, with examples up to eleventh order, and they have multidimensional extensions. One of the algorithm families has spectral like high resolution. Propagation with high order and high resolution algorithms can produce accurate results after O(10(exp 6)) periods of propagation with eight grid points per wavelength.

  11. Software-assisted serum metabolite quantification using NMR.

    PubMed

    Jung, Young-Sang; Hyeon, Jin-Seong; Hwang, Geum-Sook

    2016-08-31

    The goal of metabolomics is to analyze a whole metabolome under a given set of conditions, and accurate and reliable quantitation of metabolites is crucial. Absolute concentration is more valuable than relative concentration; however, the most commonly used method in NMR-based serum metabolic profiling, bin-based and full data point peak quantification, provides relative concentration levels of metabolites and are not reliable when metabolite peaks overlap in a spectrum. In this study, we present the software-assisted serum metabolite quantification (SASMeQ) method, which allows us to identify and quantify metabolites in NMR spectra using Chenomx software. This software uses the ERETIC2 utility from TopSpin to add a digitally synthesized peak to a spectrum. The SASMeQ method will advance NMR-based serum metabolic profiling by providing an accurate and reliable method for absolute quantification that is superior to bin-based quantification. PMID:27506360

  12. Novel accurate bacterial discrimination by MALDI-time-of-flight MS based on ribosomal proteins coding in S10-spc-alpha operon at strain level S10-GERMS.

    PubMed

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  13. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    NASA Astrophysics Data System (ADS)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  14. Quantification of bovine milk protein composition and coagulation properties using infrared spectroscopy and chemometrics: A result of collinearity among reference variables.

    PubMed

    Eskildsen, C E; Skov, T; Hansen, M S; Larsen, L B; Poulsen, N A

    2016-10-01

    Predicting protein fractions and coagulation properties in bovine milk using Fourier transform infrared (FT-IR) measurements is desirable. However, such predictions may rely on correlations with total protein content. The aim of this study was to show how correlations between total protein content, protein fractions, and coagulation properties are responsible for the successful prediction of protein fractions and rennet-induced coagulation properties in milk samples. This study comprised 832 bovine milk samples from 2 breeds (426 Holstein and 406 Jersey). Holstein samples were collected from 20 Danish dairy herds from October to December 2009; Jersey samples were collected from 22 Danish dairy herds from February to April 2010. All samples were from conventional herds and taken while cows were housed. The results showed that κ-CN, αS1-CN, αS1-CN with 8 phosphorylated groups attached (αS1-CN 8P), and curd firming rate could be predicted from FT-IR measurements of the milk samples (with coefficients of determination between 0.66 and 0.71). However, the success of these FT-IR-based predictions was based on indirect relationships with total protein content. Hence, the FT-IR predictions relied on covariance structures with total protein content rather than absorption bands directly associated with the protein fractions and coagulation properties. If covariance structures between the protein fractions, coagulation properties, and total protein content used to calibrate partial least squares models were not conserved in future samples, these samples would show incorrect predictions of the protein fractions and coagulation properties. We demonstrated this using samples from 1 breed to calibrate and samples from the other breed to validate partial least squares models for β-CN. The 2 breeds had different covariance structures between β-CN and total protein content, and the validation samples yielded incorrect predictions. This finding may limit the usefulness of FT

  15. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating

    NASA Astrophysics Data System (ADS)

    Valbuena, Alejandro; Mateu, Mauricio G.

    2015-09-01

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics (``breathing'') of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications

  16. Quantification and modification of the equilibrium dynamics and mechanics of a viral capsid lattice self-assembled as a protein nanocoating.

    PubMed

    Valbuena, Alejandro; Mateu, Mauricio G

    2015-09-28

    Self-assembling, protein-based bidimensional lattices are being developed as functionalizable, highly ordered biocoatings for multiple applications in nanotechnology and nanomedicine. Unfortunately, protein assemblies are soft materials that may be too sensitive to mechanical disruption, and their intrinsic conformational dynamism may also influence their applicability. Thus, it may be critically important to characterize, understand and manipulate the mechanical features and dynamic behavior of protein assemblies in order to improve their suitability as nanomaterials. In this study, the capsid protein of the human immunodeficiency virus was induced to self-assemble as a continuous, single layered, ordered nanocoating onto an inorganic substrate. Atomic force microscopy (AFM) was used to quantify the mechanical behavior and the equilibrium dynamics ("breathing") of this virus-based, self-assembled protein lattice in close to physiological conditions. The results uniquely provided: (i) evidence that AFM can be used to directly visualize in real time and quantify slow breathing motions leading to dynamic disorder in protein nanocoatings and viral capsid lattices; (ii) characterization of the dynamics and mechanics of a viral capsid lattice and protein-based nanocoating, including flexibility, mechanical strength and remarkable self-repair capacity after mechanical damage; (iii) proof of principle that chemical additives can modify the dynamics and mechanics of a viral capsid lattice or protein-based nanocoating, and improve their applied potential by increasing their mechanical strength and elasticity. We discuss the implications for the development of mechanically resistant and compliant biocoatings precisely organized at the nanoscale, and of novel antiviral agents acting on fundamental physical properties of viruses.

  17. Quantification of Secondary Metabolites.

    PubMed

    2016-01-01

    Plants are a rich source of secondary metabolites that have medicinal and aromatic properties. Secondary metabolites such as alkaloids, iridoids and phenolics generally produced by plants for their defence mechanisms have been implicated in the therapeutic properties of most medicinal plants. Hence, quantification of these metabolites will aid to discover new and effective drugs from plant sources and also to scientifically validate the existing traditional practices. Quantification of large group of phytochemicals such as phenolics and flavonoids is quantified in this context.

  18. Detection and quantification of bacterial spoilage in milk and pork meat using MALDI-TOF-MS and multivariate analysis.

    PubMed

    Nicolaou, Nicoletta; Xu, Yun; Goodacre, Royston

    2012-07-17

    Microbiological safety is one of the cornerstones of quality control in the food industry. Identification and quantification of spoilage bacteria in pasteurized milk and meat in the food industry currently relies on accurate and sensitive yet time-consuming techniques which give retrospective values for microbial contamination. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a proven technique in the field of protein and peptide identification and quantification, may be a valuable alternative approach for the rapid assessment of microbial spoilage. In this work we therefore developed MALDI-TOF-MS as a novel analytical approach for the assessment of food that when combined with chemometrics allows for the detection and quantification of milk and pork meat spoilage bacteria. To develop this approach, natural spoilage of pasteurized milk and raw pork meat samples incubated at 15 °C and at room temperature, respectively, was conducted. Samples were collected for MALDI-TOF-MS analysis (which took 4 min per sample) at regular time intervals throughout the spoilage process, with concurrent calculation and documentation of reference total viable counts using traditional microbiological methods (these took 2 days). Multivariate statistical techniques such as principal component discriminant function analysis, canonical correlation analysis, partial least-squares (PLS) regression, and kernel PLS (KPLS) were used to analyze the data. The results from MALDI-TOF-MS combined with PLS or KPLS gave excellent bacterial quantification results for both milk and meat spoilage, and typical root mean squared errors for prediction in test spectra were between 0.53 and 0.79 log unit. Overall these novel findings strongly indicate that MALDI-TOF-MS when combined with chemometric approaches would be a useful adjunct for routine use in the milk and meat industry as a fast and accurate viable bacterial detection and quantification method.

  19. Accurate quantum chemical calculations

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Langhoff, Stephen R.; Taylor, Peter R.

    1989-01-01

    An important goal of quantum chemical calculations is to provide an understanding of chemical bonding and molecular electronic structure. A second goal, the prediction of energy differences to chemical accuracy, has been much harder to attain. First, the computational resources required to achieve such accuracy are very large, and second, it is not straightforward to demonstrate that an apparently accurate result, in terms of agreement with experiment, does not result from a cancellation of errors. Recent advances in electronic structure methodology, coupled with the power of vector supercomputers, have made it possible to solve a number of electronic structure problems exactly using the full configuration interaction (FCI) method within a subspace of the complete Hilbert space. These exact results can be used to benchmark approximate techniques that are applicable to a wider range of chemical and physical problems. The methodology of many-electron quantum chemistry is reviewed. Methods are considered in detail for performing FCI calculations. The application of FCI methods to several three-electron problems in molecular physics are discussed. A number of benchmark applications of FCI wave functions are described. Atomic basis sets and the development of improved methods for handling very large basis sets are discussed: these are then applied to a number of chemical and spectroscopic problems; to transition metals; and to problems involving potential energy surfaces. Although the experiences described give considerable grounds for optimism about the general ability to perform accurate calculations, there are several problems that have proved less tractable, at least with current computer resources, and these and possible solutions are discussed.

  20. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

    PubMed Central

    Osinalde, Nerea; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Blagoev, Blagoy; Kratchmarova, Irina

    2015-01-01

    This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129. PMID:26425665

  1. Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein.

    PubMed Central

    Kruckeberg, A L; Ye, L; Berden, J A; van Dam, K

    1999-01-01

    The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10(5) Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 degrees C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose. PMID:10191260

  2. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis.

  3. Accurate Optical Reference Catalogs

    NASA Astrophysics Data System (ADS)

    Zacharias, N.

    2006-08-01

    Current and near future all-sky astrometric catalogs on the ICRF are reviewed with the emphasis on reference star data at optical wavelengths for user applications. The standard error of a Hipparcos Catalogue star position is now about 15 mas per coordinate. For the Tycho-2 data it is typically 20 to 100 mas, depending on magnitude. The USNO CCD Astrograph Catalog (UCAC) observing program was completed in 2004 and reductions toward the final UCAC3 release are in progress. This all-sky reference catalogue will have positional errors of 15 to 70 mas for stars in the 10 to 16 mag range, with a high degree of completeness. Proper motions for the about 60 million UCAC stars will be derived by combining UCAC astrometry with available early epoch data, including yet unpublished scans of the complete set of AGK2, Hamburg Zone astrograph and USNO Black Birch programs. Accurate positional and proper motion data are combined in the Naval Observatory Merged Astrometric Dataset (NOMAD) which includes Hipparcos, Tycho-2, UCAC2, USNO-B1, NPM+SPM plate scan data for astrometry, and is supplemented by multi-band optical photometry as well as 2MASS near infrared photometry. The Milli-Arcsecond Pathfinder Survey (MAPS) mission is currently being planned at USNO. This is a micro-satellite to obtain 1 mas positions, parallaxes, and 1 mas/yr proper motions for all bright stars down to about 15th magnitude. This program will be supplemented by a ground-based program to reach 18th magnitude on the 5 mas level.

  4. Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²⁺ concentration in boar spermatozoa during cryopreservation.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Johannisson, A; Eriksson, I; Wallgren, M; Bergqvist, A S

    2012-01-01

    Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation. PMID:22541541

  5. Neuroanatomical localization and quantification of amyloid precursor protein mRNA by in situ hybridization in the brains of normal, aneuploid, and lesioned mice

    SciTech Connect

    Bendotti, C.; Forloni, G.L.; Morgan, R.A.; O'Hara, B.F.; Oster-Granite, M.L.; Reeves, R.H.; Gearhart, J.D.; Coyle, J.T. )

    1988-05-01

    Amyloid precursor protein mRNA was localized in frozen sections from normal and experimentally lesioned adult mouse brain and from normal and aneuploid fetal mouse brain by in situ hybridization with a {sup 35}S-labeled mouse cDNA probe. The highest levels of hybridization in adult brain were associated with neurons, primarily in telencephalic structures. The dense labeling associated with hippocampal pyramidal cells was reduced significantly when the cells were eliminated by injection of the neurotoxin ibotenic acid but was not affected when electrolytic lesions were placed in the medial septum. Since the gene encoding amyloid precursor protein has been localized to mouse chromosome 16, the authors also examined the expression of this gene in the brains of mouse embryos with trisomy 16 and trisomy 19 at 15 days of gestation. RNA gel blot analysis and in situ hybridization showed a marked increase in amyloid precursor protein mRNA in the trisomy 16 mouse head and brain when compared with euploid littermates or with trisomy 19 mice.

  6. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-10-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  7. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues.

    PubMed

    Steiner, Carine; Tille, Jean-Christophe; Lamerz, Jens; Kux van Geijtenbeek, Sabine; McKee, Thomas A; Venturi, Miro; Rubbia-Brandt, Laura; Hochstrasser, Denis; Cutler, Paul; Lescuyer, Pierre; Ducret, Axel

    2015-10-01

    The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

  8. Separation and quantification of double- and triple-layered rotavirus-like particles by CZE.

    PubMed

    Castro-Acosta, Ricardo M; Revilla, Alma L; Ramírez, Octavio T; Palomares, Laura A

    2010-04-01

    Virus-like particles have been successfully used as safe vaccines, as their structure is identical to their native counterparts but devoid of the viral genetic material. However, production of these complex structures is not easy, as recombinant proteins must assemble into virus-like particles. Techniques to differentiate assembled and soluble proteins, as well as assembly intermediaries often present in a sample, are required. An example of complex virus-like particles mixture occurs when rotavirus proteins are recombinantly expressed. Rotavirus-like particles (RLP) can be single (sl), double (dl), or triple layered (tl). The use of RLP preparations as vaccines requires their complete characterization, including separation and quantification of each RLP in a sample. In this work, CZE was evaluated for the separation and quantification of dl and triple-layered rotavirus-like particles (tlRLP). A fused-silica capillary with a deoxycholate running buffer efficiently separated dl and tlRLP in RLP preparations, as they migrated in two discrete peaks with electrophoretic mobilities of 1.24+/-0.04 and 2.95+/-0.03 Ti, respectively. Standard curves for dl and tlRLP were generated, and the response was linearly proportional to analyte concentration. The methodology developed was quantitative, specific, accurate, precise, and reproducible. CZE allowed the quantitative characterization of RLP preparations, which is required for evaluation of immunogens, for process development, and for quality control protocols.

  9. Quantification of nonclassicality

    NASA Astrophysics Data System (ADS)

    Gehrke, C.; Sperling, J.; Vogel, W.

    2012-11-01

    To quantify single-mode nonclassicality, we start from an operational approach. A positive semidefinite observable is introduced to describe a measurement setup. The quantification is based on the negativity of the normally ordered version of this observable. Perfect operational quantumness corresponds to the quantum-noise-free measurement of the chosen observable. Surprisingly, even moderately squeezed states may exhibit perfect quantumness for a properly designed measurement. The quantification is also considered from an axiomatic viewpoint, based on the algebraic structure of the quantum states and the quantum superposition principle. Basic conclusions from both approaches are consistent with this fundamental principle of the quantum world.

  10. Quantification of Secondary Metabolites.

    PubMed

    2016-01-01

    Plants are a rich source of secondary metabolites that have medicinal and aromatic properties. Secondary metabolites such as alkaloids, iridoids and phenolics generally produced by plants for their defence mechanisms have been implicated in the therapeutic properties of most medicinal plants. Hence, quantification of these metabolites will aid to discover new and effective drugs from plant sources and also to scientifically validate the existing traditional practices. Quantification of large group of phytochemicals such as phenolics and flavonoids is quantified in this context. PMID:26939265

  11. Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis.

    PubMed

    Morgenstern, Josefine; Busch, Markus; Baumann, Pascal; Hubbuch, Jürgen

    2016-09-01

    PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis. PMID:27521256

  12. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

    PubMed

    Yilmaz, Ozlem; Prat, Francisco; Ibáñez, A Jose; Köksoy, Sadi; Amano, Haruna; Sullivan, Craig V

    2016-01-01

    Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment. PMID:26643259

  13. Multiple vitellogenins and product yolk proteins in European sea bass (Dicentrarchus labrax): Molecular characterization, quantification in plasma, liver and ovary, and maturational proteolysis.

    PubMed

    Yilmaz, Ozlem; Prat, Francisco; Ibáñez, A Jose; Köksoy, Sadi; Amano, Haruna; Sullivan, Craig V

    2016-01-01

    Three complete vitellogenin (Vtg) polypeptides of European sea bass (Dicentrarchus labrax), an acanthomorph teleost spawning pelagic eggs in seawater, were deduced from cDNA and identified as VtgAa, VtgAb and VtgC based on current Vtg nomenclature and phylogeny. Label free quantitative mass spectrometry verified the presence of the three sea bass Vtgs or their product yolk proteins (YPs) in liver, plasma and ovary of postvitellogenic females. As evidenced by normalized spectral counts, VtgAb-derived protein was 2- to 5-fold more abundant, depending on sample type, than for VtgAa, while VtgC-derived protein was less abundant, albeit only 3-fold lower than for VtgAb in the ovary. Western blotting with Vtg type-specific antisera raised against corresponding gray mullet (Mugil cephalus) lipovitellins (Lvs) detected all three types of sea bass Vtg in the blood plasma of gravid females and/or estrogenized males and showed that all three forms of sea bass Lv undergo limited partial degradation during oocyte maturation. The comparatively high levels of VtgC-derived YPs in fully-grown oocytes and the maturational proteolysis of all three types of Lv differ from what has been reported for other teleosts spawning pelagic eggs in seawater but are similar to recent findings for two species of North American Moronidae, the striped bass (Morone saxatilis) and white perch (Morone americana), which spawn pelagic and demersal eggs, respectively in fresh water. Together with the high Vtg sequence homologies and virtually identical structural features of each type of Vtg between species, these findings indicate that the moronid multiple Vtg systems do not substantially vary with reproductive environment.

  14. Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis.

    PubMed

    Morgenstern, Josefine; Busch, Markus; Baumann, Pascal; Hubbuch, Jürgen

    2016-09-01

    PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis.

  15. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

    PubMed

    Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.

  16. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    PubMed

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation.

  17. A simplified hydroethidine method for fast and accurate detection of superoxide production in isolated mitochondria.

    PubMed

    Back, Patricia; Matthijssens, Filip; Vanfleteren, Jacques R; Braeckman, Bart P

    2012-04-01

    Because superoxide is involved in various physiological processes, many efforts have been made to improve its accurate quantification. We optimized and validated a superoxide-specific and -sensitive detection method. The protocol is based on fluorescence detection of the superoxide-specific hydroethidine (HE) oxidation product, 2-hydroxyethidium. We established a method for the quantification of superoxide production in isolated mitochondria without the need for acetone extraction and purification chromatography as described in previous studies.

  18. Quantification of Wheat Grain Arabinoxylans Using a Phloroglucinol Colorimetric Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabinoxylans (AX) play a critical role in end-use quality and nutrition of wheat (Triticum aestivum L.). An efficient, accurate method of AX quantification is desirable as AX plays an important role in processing, end use quality and human health. The objective of this work was to evaluate a stand...

  19. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    USGS Publications Warehouse

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  20. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    PubMed

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea. PMID:27154315

  1. Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid.

    PubMed

    Kumaresan, A; González, R; Johannisson, A; Berqvist, A-S

    2014-11-01

    Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorter time of exposure to capacitating conditions. Washed, frozen-thawed boar spermatozoa were incubated separately with pre-ovulatory isthmic oviductal fluid (EODF), post-ovulatory ODF (MODF), capacitation medium (CM), and noncapacitating medium (NCM) for 60 minutes. Aliquots of spermatozoa were taken at 0, 5, 15, 30, and 60 minutes during incubation and sperm kinematics, intracellular calcium [Ca2(+)]i content, and protein tyrosine phosphorylation (PTP) were studied. The proportion of motile spermatozoa increased significantly after 5 minutes of incubation with EODF. A similar increase was not observed in the other groups. During the initial 5 minutes of incubation, the proportion of spermatozoa with high [Ca(2+)]i decreased significantly in all four groups. The proportion of tyrosine phosphorylated spermatozoa increased from 6.49 ± 1.93% to 15.42 ± 3.58% and 18.41 ± 1.57% in EODF and MODF groups, respectively, at 5 minutes of incubation. Neither CM nor NCM elicited any immediate effect on PTP in spermatozoa. There was a positive and significant correlation between [Ca(2+)]i and sperm motility (P = 0.009). It may be concluded that frozen-thawed boar spermatozoa undergo capacitation-associated changes after a relatively short exposure to EODF, and there are some subpopulations of spermatozoa that undergo PTP despite possessing low [Ca(2+)]i. PMID:25175760

  2. Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

    PubMed

    Gjoka, Xhorxhi; Schofield, Mark; Cvetkovic, Aleksandar; Gantier, Rene

    2014-12-01

    Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford.

  3. Quantificational logic of context

    SciTech Connect

    Buvac, Sasa

    1996-12-31

    In this paper we extend the Propositional Logic of Context, to the quantificational (predicate calculus) case. This extension is important in the declarative representation of knowledge for two reasons. Firstly, since contexts are objects in the semantics which can be denoted by terms in the language and which can be quantified over, the extension enables us to express arbitrary first-order properties of contexts. Secondly, since the extended language is no longer only propositional, we can express that an arbitrary predicate calculus formula is true in a context. The paper describes the syntax and the semantics of a quantificational language of context, gives a Hilbert style formal system, and outlines a proof of the system`s completeness.

  4. Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS.

    PubMed

    Grebe, Mechthild; Pröfrock, Daniel; Kakuschke, Antje; Broekaert, Jose A C; Prange, Andreas

    2011-02-01

    Harbour seals (Phoca vitulina) are bio-indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status represents a further important parameter for this assessment. The health status of seals is a complex and vague term, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L(-1) (sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L(-1), confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf

  5. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

    PubMed

    Zhu, Xiaoyang; Li, Xueping; Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

    2012-01-01

    Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.

  6. Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry.

    PubMed

    Kim, Jun Seok; Ahn, Hee-Sung; Cho, Soo Min; Lee, Ji Eun; Kim, YoungSoo; Lee, Cheolju

    2014-08-20

    Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ(1-5), Aβ(6-16), Aβ(17-28) and Aβ(29-40(42)) of either Aβ(1-40) or Aβ(1-42). Among those peptides, Aβ(17-28) was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL(-1) with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL(-1) of Aβ(1-40). Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker. PMID:25086887

  7. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    PubMed

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  8. Practical quantification of necrosis in histological whole-slide images.

    PubMed

    Homeyer, André; Schenk, Andrea; Arlt, Janine; Dahmen, Uta; Dirsch, Olaf; Hahn, Horst K

    2013-06-01

    Since the histological quantification of necrosis is a common task in medical research and practice, we evaluate different image analysis methods for quantifying necrosis in whole-slide images. In a practical usage scenario, we assess the impact of different classification algorithms and feature sets on both accuracy and computation time. We show how a well-chosen combination of multiresolution features and an efficient postprocessing step enables the accurate quantification necrosis in gigapixel images in less than a minute. The results are general enough to be applied to other areas of histological image analysis as well. PMID:23796718

  9. SERS Quantification of Entacapone Isomers

    NASA Astrophysics Data System (ADS)

    Marković, Marina; Biljan, Tomislav

    2010-08-01

    Raman spectroscopy, due to its non-destructive character and speed, has found widespread use in pharmaceutical applications [1]. It is also being used for quantifying various isomer mixtures, best known being the quantification of xylene isomers [2-3]. Solid-state isomer quantification of entacapone was earlier reported [4]. Here, we report quantification of isomer mixture of an active pharmaceutical substance, in solution, by SERS.

  10. Automated renal histopathology: digital extraction and quantification of renal pathology

    NASA Astrophysics Data System (ADS)

    Sarder, Pinaki; Ginley, Brandon; Tomaszewski, John E.

    2016-03-01

    The branch of pathology concerned with excess blood serum proteins being excreted in the urine pays particular attention to the glomerulus, a small intertwined bunch of capillaries located at the beginning of the nephron. Normal glomeruli allow moderate amount of blood proteins to be filtered; proteinuric glomeruli allow large amount of blood proteins to be filtered. Diagnosis of proteinuric diseases requires time intensive manual examination of the structural compartments of the glomerulus from renal biopsies. Pathological examination includes cellularity of individual compartments, Bowman's and luminal space segmentation, cellular morphology, glomerular volume, capillary morphology, and more. Long examination times may lead to increased diagnosis time and/or lead to reduced precision of the diagnostic process. Automatic quantification holds strong potential to reduce renal diagnostic time. We have developed a computational pipeline capable of automatically segmenting relevant features from renal biopsies. Our method first segments glomerular compartments from renal biopsies by isolating regions with high nuclear density. Gabor texture segmentation is used to accurately define glomerular boundaries. Bowman's and luminal spaces are segmented using morphological operators. Nuclei structures are segmented using color deconvolution, morphological processing, and bottleneck detection. Average computation time of feature extraction for a typical biopsy, comprising of ~12 glomeruli, is ˜69 s using an Intel(R) Core(TM) i7-4790 CPU, and is ~65X faster than manual processing. Using images from rat renal tissue samples, automatic glomerular structural feature estimation was reproducibly demonstrated for 15 biopsy images, which contained 148 individual glomeruli images. The proposed method holds immense potential to enhance information available while making clinical diagnoses.

  11. nagnag: Identification and quantification of NAGNAG alternative splicing using RNA-Seq data.

    PubMed

    Yan, Xiaoyan; Sablok, Gaurav; Feng, Gang; Ma, Jiaxin; Zhao, Hongwei; Sun, Xiaoyong

    2015-07-01

    Regulation of proteome diversity by alternative splicing has been widely demonstrated in plants and animals. NAGNAG splicing, which was recently defined as a tissue specific event, results in the production of two distinct isoforms that are distinguished by three nucleotides (NAG) as a consequence of the intron proximal or distal to the splice site. Since the NAGNAG mechanism is not well characterized, tools for the identification and quantification of NAGNAG splicing events remain under-developed. Here we report nagnag, an R-based NAGNAG splicing detection tool, which accurately identifies and quantifies NAGNAG splicing events using RNA-Seq. Overall, nagnag produces user-friendly visualization reports and highlights differences between the DNA/RNA/protein across the identified isoforms of the reported gene. The package is available on https://sourceforge.net/projects/nagnag/files/; or http://genome.sdau.edu.cn/research/software/nagnag.html. PMID:26028313

  12. Fluorophores, environments and quantification techniques in the analysis of transmembrane helix interaction us