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QUANTITATIVE for clinical diagnostics quantification: the quantity precision required for the Maria Colombo of the biomarker is detection of low abundance & Véronique determined by comparing biomarkers, further pushing Mainfroid the MS signal generated by a the limits of detection and detail how tryptic peptide of the biomarker quantification of protein accurate peptide with the signal from a peptide biomarkers in complex samples. quantification equivalent, heavy-isotope Fig. 1. shows the MS signals labelled and spiked into the obtained for two 15-mers supports the sample. Moreover, it is well peptides selected from transfer of MS- adapted to multiplexing and sharing more than 95% identity, based quantitative to high-throughput assays. peptides differing from 1 amino- proteomics to the MRM uses the strategy based acid used for protein variant clinic on stable isotope-labelled analysis. standards for absolute Sample preparation also quantification. deserves particular attention, espite the identification because biomarkers may and validation of MS-based quantitative be extremely diluted, while D numerous potential proteomics the sample matrix may biomarkers for medical Because significantly interfere with diagnostics, only a few of them relies on the ability to detect biomarker detection. Depletion, actually progressed towards small changes in protein fractionation and improved clinical use. abundance, all steps of the liquid chromatography (LC) ELISA remains the gold MRM process must be under technologies are therefore standard to quantify proteins, tight control, including continuously evolving. however it is often limited instrumentation, sample because of the lack of availability preparation and biomarker Isotope-labelled of antibodies with high analysis and together, their peptides specificity and sensitivity. imprecision cannot exceed the Stable isotope-labelled peptides The development of more biomarker variability in test keep a central position. The direct detection methods aims samples 3, 4. Accordingly, major quantification of protein at solving this intrinsic issue technological improvements biomarkers using MRM is linked with immunoassays. were done at each step of the indeed only achievable if the Indeed, over the past decade, MRM process. peptides spiked into the sample (MS)-based High-resolution instruments perfectly match the endogenous quantitative proteomics has are continuously developed, biomarker equivalent. While gained considerable interest 1, providing the reliability and very complex peptides can be as it is the most promising technology for allowing amino acid-based biomarker quantification. As such, multiple reaction monitoring (MRM) is considered today as a major technology for accurate quantitative proteomics 2.

Absolute biomarker Fig. 1. UPLC MS-MS signal quantification of 15-mer peptides which The development of strategies sequences present one amino- based on stable isotope-labelled acid variant. Peptides selected standards makes it possible to use from protein showing more MRM for absolute biomarker than 95% homology www.scientistlive.com 60 BIOTECHNOLOGY

REFERENCES: 1 Wasinger V.C. et al. (2013). Current status and advances in quantitative proteomic mass spectrometry. Int. J. Proteomics 2013: 180605. 2 Gianazza E. et al. (2014). The selected reaction monitoring/ multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases. Expert Rev. Proteomics 11(6): 771-788. 3 Van den Broek I. et al. (2015). Quantifying protein measurands by peptide measurements: where do errors arise? J. Proteome Res. 14)2): 928-942. 4 Arnold S.L. et al. (2015). Impact of Sample Matrix on Accuracy of Peptide Quantification: Assessment of Calibrator and Internal Fig. 2. UPLC signal of tagged- more sensitive than the classical Standard Selection and Method Validation. Anal. Chem. peptides (black line) and after AAA. 5b03004. tryptic digestion (red line) 5 Louwagie, M. et al. (2012). Introducing AAA-MS, a rapid and Tagged peptides sensitive method for amino acid analysis using isotope dilution synthesised by expert chemists, Alternative quantification can and high-resolution mass spectrometry. J. Proteome Res. 11: the limitation remains on how be made using tagged peptides. 3929-3936. accurately they can be quantified, In this case the absolute as they are intended to be used quantification is based on the as internal calibrators. Amino inherent spectral properties of acid analysis (AAA), which is the the proprietary tag. The Quant- to the tag mass is indicative of traditional peptide quantification Tag is coupled to the C-term trypsin digestion. method, offers limited of the peptide via an Arginine reproducibility and precision. (R) or Lysine (K) residue and Accurate peptide As such, the seminal publication can be released by trypsin quantification from M. Louwagie et al. 5, which digestion. With these technologies, fully describes a rapid, sensitive and The precise molecular calibrated peptide standards can reliable quantification method mass of the tag can be used in now be offered, highly improving for peptides and proteins, offers assessing the cleavage efficacy, the global analytical performance an extremely important and and hence in setting the optimal of MRM. Fig. 3. shows the promising achievement in the trypsinisation conditions of a similarity of signal obtained with peptide field. sample using UPLC-MS-MS the two different quantifications This method, AAA-MS, methods. To do so, the tagged methods using tag spectral avoids using derivatisation and peptide must be spiked into the properties of mass spectroscopy chromatographic separation sample prior to trypsin digestion. amino-acid analysis. of amino acids, and is based Fig. 2. shows the UPLC It therefore appears that we on a rapid microwave-assisted signals obtained with a tagged do have everything in hand to acidic hydrolysis followed by peptide before and after cleavage. achieve the goal of transposing high-resolution MS analysis of The Quant-Tag could be used as MRM into clinics, offering hope amino acids (ESI-based). This a reporter of the trypsin digestion that MS-based protein biomarker technology proved to be 100-fold efficacy: a peak corresponding detection and quantification will become effective in routine assays in a very near future.

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Fig. 3. UPLC-MS-MS signals comparison from peptide quantity obtained after tryptic digestion of tagged-peptide (spectral quantification) (A); non-tagged control peptide Maria Colombo & (AAA-MS quantification) Véronique Mainfroid are that has followed the tryptic with Eurogentec. digestion process (B) and non- www.eurogentec.com tagged control peptide (C) www.scientistlive.com