Fig. 3. Workflow of mass spectrometry (MS)-based quantitation of endogenous peptides. Romanova et al. Curr Opin Chem Biol 2013;17:801-808, with permission of Elsevier [51]. Quantitation of endogenous peptides based on MS is generally achieved either by stable isotope labeling (A) or label-free approaches (B). In stable isotope labeling, the endogenous peptides in two different samples are labeled with either a light stable isotope or a heavy stable isotope, and the two samples are then combined and analyzed together. The relative levels of the peptides are calculated by the difference in the MS peaks (peak intensity or peak area) of the two samples. In label-free quantitation, each sample is prepared separately and then subjected to individual liquid chromatography (LC)-MS or LC–tandem mass spectrometry (MS/MS) runs, followed by comparisons of either the mass spectral peak intensities of the detected peptides or the total number of MS/MS spectra identified for a peptide, called spectral counting.