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p. 28 / OVERVIEW ON PEPTIDES
Accurate peptide quantification supports the transfer of MS-based quantitative proteomics to the clinic
VÉRONIQUE MAINFROID
Proteomics Product Manager, Eurogentec S.A.

KEYWORDS: Quantitative proteomic, AAA-MS, peptide accurate quantification, SIL-peptide, MRM.

QUANTITATIVE PROTEOMIC FOR CLINIC DIAGNOSTIC
Despite the identification and validation of numerous potential biomarkers for medical diagnostic, only few of them actually progressed towards clinical use. ELISA remaining the gold standard to quantify proteins, it is often limited to the lack of availability of antibodies with high specificity. The development of more direct detection methods aims at solving this intrinsic issue linked with immunoassays. Indeed, over the last decade, mass spectrometry (MS)-based quantitative proteomics has gained considerable interest (1), as it is the most promising technology allowing amino acid-based biomarker quantification. As such, Multiple Reaction Monitoring (MRM) is considered today as a major technology for accurate quantitative proteomics (2). Moreover, it is well adapted to multiplexing and to high-throughput assays. MRM uses the strategy based on stable isotope-labeled standards for absolute quantification.

MASS SPECTROMETRY (MS)-BASED QUANTITATIVE PROTEOMICS

Because quantitative proteomics relies on the ability to detect small changes in protein abundance, all steps of the MRM process must be under tight control, including instrumentation, sample preparation and biomarker analysis and together, their imprecision cannot exceed the biomarker variability in test samples (3, 4). Accordingly, major technological improvements were done at each step of the MRM process.
High-resolution instruments are continuously developed, providing the reliability and precision required for the detection of low abundance biomarkers, further pushing the limits of detection and quantification of protein biomarkers in complex samples.
Sample preparation also deserves particular attention, because biomarkers may be extremely diluted, while the sample matrix may significantly interfere with biomarker detection. Depletion, fractionation and improved liquid chromatography technologies are therefore continuously evolving.
Stable isotopes-labeled peptides keep a central position. The quantification of protein biomarkers using MRM is indeed only achievable if the peptides spiked into the sample do perfectly match the endogenous biomarker equivalent. While very complex peptides can be synthesized by expert chemists, the limitation remains on how accurately they can be quantified, as they are intended to be used as internal calibrators. Amino Acid Analysis (AAA), which is the traditional peptide quantification method, offers limited reproducibility and precision. As such, the seminal publication from M. Louwagie et al. (5), which describes a rapid, sensitive and reliable quantification method for peptides and proteins, offers an extremely important and promising achievement in the peptide field. This method, AAA-MS, avoids using derivatization and chromatographic separation of amino acids, and is based on a rapid microwave-assisted acidic hydrolysis followed by high-resolution MS analysis of amino acids (ESI-based). This technology proved to be 100-fold more sensitive than the classical AAA.

ACCURATE PEPTIDE QUANTIFICATION

With this AAA-MS technology, one can now offer fully calibrated peptide...In order to continue reading this article please register to our website – registration is for free and no fees will be applied afterwards to download contents.

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