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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


Fig. 2. UPLC signal of tagged- peptides (black line) and after tryptic digestion (red line)


synthesised 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.


Tis method, AAA-MS,


avoids using derivatisation 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). Tis technology proved to be 100-fold


more sensitive than the classical AAA.


Tagged peptides Alternative quantification can be made using tagged peptides. In this case the absolute quantification is based on the inherent spectral properties of the proprietary tag. Te Quant- Tag is coupled to the C-term of the peptide via an Arginine (R) or Lysine (K) residue and can be released by trypsin digestion. Te precise molecular mass of the tag can be used in assessing the cleavage efficacy, and hence in setting the optimal trypsinisation conditions of a sample using UPLC-MS-MS methods. To do so, the tagged peptide must be spiked into the sample prior to trypsin digestion. Fig. 2. shows the UPLC signals obtained with a tagged peptide before and after cleavage. Te Quant-Tag could be used as a reporter of the trypsin digestion efficacy: a peak corresponding


Arnold S.L. et al. (2015). Impact of Sample Matrix on Accuracy


of Peptide Quantification: Assessment of Calibrator and Internal Standard Selection and Method Validation. Anal. Chem. 5b03004. 5


Louwagie, M. et al. (2012). Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry. J. Proteome Res. 11: 3929-3936.


to the tag mass is indicative of trypsin digestion.


Accurate peptide quantification With these technologies, fully calibrated peptide standards can now be offered, highly improving the global analytical performance of MRM. Fig. 3. shows the similarity of signal obtained with the two different quantifications methods using tag spectral properties of mass spectroscopy amino-acid analysis. It therefore appears that we


do have everything in hand to achieve the goal of transposing MRM into clinics, offering hope that MS-based protein biomarker detection and quantification will become effective in routine assays in a very near future.


For more information ✔ at www.scientistlive.com/eurolab


Fig. 3. UPLC-MS-MS signals comparison from peptide quantity obtained after tryptic digestion of tagged-peptide (spectral quantification) (A); non-tagged control peptide (AAA-MS quantification) that has followed the tryptic digestion process (B) and non- tagged control peptide (C)


www.scientistlive.com


Maria Colombo & Véronique Mainfroid are with Eurogentec. www.eurogentec.com


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