Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration
LeBlanc, A.; Michaud, S.A.; Percy, A.J.; et al.
Toward enhanced depth and precision/accuracy of multiplexed protein quantitation, LeBlanc et al. developed a new calibration approach utilizing two variations of stable isotope-labeled tryptic peptides (referred to as SIS 1 and SIS 2). The SIS 1 peptides have a C-terminal 13C/15N-label, while SIS 2 has an internal 13C/15N-labeled residue in addition to a C-terminal label. Procedurally, external curves and QCs are prepared in a surrogate matrix using a constant amount of SIS 1 and a variable amount of SIS 2. This double-SIS-peptide approach overcomes matrix-induced interference from endogenous peptides and, compared to conventional quantitative approaches, provides superior figures of merit for improved method development and assay validation.
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