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.