A typical LC-MRM/MS experiment. In a standard absolute quantitative experiment, proteins are first extracted from the biological sample and then denatured, reduced, and enzymatically digested (usually with trypsin). The digest is spiked with a known amount of stable-isotope–labeled standard (SIS) peptide corresponding to the targeted peptides from the targeted proteins. The mixture is then submitted for analysis by MS, where the endogenous and isotopically labeled peptides are explicitly identified by retention time and peak shape, precursor and product ion mass-to-charge ratio, and fragment ion ratios. In the mass spectrometer, specific precursor ions are selected in the first mass analyzer according to their mass-to-charge ratio; they are then fragmented by collision-induced dissociation in the collision cell and mass filtered in the second mass analyzer; only the targeted fragment ion reaches the detector. Finally, comparison of the signal from the endogenous peptide and the known amount of isotopically labeled internal standard provides the absolute quantitation.