Dataset Information

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

ABSTRACT: Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies.

SUBMITTER: Huillet C

PROVIDER: S-EPMC3277748 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Huillet Céline C Adrait Annie A Lebert Dorothée D Picard Guillaume G Trauchessec Mathieu M Louwagie Mathilde M Dupuis Alain A Hittinger Luc L Ghaleh Bijan B Le Corvoisier Philippe P Jaquinod Michel M Garin Jérôme J Bruley Christophe C Brun Virginie V

Molecular & cellular proteomics : MCP 20111111 2

Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete tr ...[more]

PMID: 22080464

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Project description:For many research questions in modern molecular and systems biology, information about absolute protein quantities is imperative. This information includes, for example, kinetic modeling of processes, protein turnover determinations, stoichiometric investigations of protein complexes, or quantitative comparisons of different proteins within one sample or across samples. To date, the vast majority of proteomic studies are limited to providing relative quantitative comparisons of protein levels between limited numbers of samples. Here we describe and demonstrate the utility of a targeting MS technique for the estimation of absolute protein abundance in unlabeled and nonfractionated cell lysates. The method is based on selected reaction monitoring (SRM) mass spectrometry and the "best flyer" hypothesis, which assumes that the specific MS signal intensity of the most intense tryptic peptides per protein is approximately constant throughout a whole proteome. SRM-targeted best flyer peptides were selected for each protein from the peptide precursor ion signal intensities from directed MS data. The most intense transitions per peptide were selected from full MS/MS scans of crude synthetic analogs. We used Monte Carlo cross-validation to systematically investigate the accuracy of the technique as a function of the number of measured best flyer peptides and the number of SRM transitions per peptide. We found that a linear model based on the two most intense transitions of the three best flying peptides per proteins (TopPep3/TopTra2) generated optimal results with a cross-correlated mean fold error of 1.8 and a squared Pearson coefficient R(2) of 0.88. Applying the optimized model to lysates of the microbe Leptospira interrogans, we detected significant protein abundance changes of 39 target proteins upon antibiotic treatment, which correlate well with literature values. The described method is generally applicable and exploits the inherent performance advantages of SRM, such as high sensitivity, selectivity, reproducibility, and dynamic range, and estimates absolute protein concentrations of selected proteins at minimized costs.

Dataset Information

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Publications

Accurate quantification of cardiovascular biomarkers in serum using Protein Standard Absolute Quantification (PSAQ™) and selected reaction monitoring.

Similar Datasets

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