Analyzing a complex sample by MS/MS using isotopically-labeled standards

A method for analyzing a peptide-containing sample by mass spectrometry, comprising:

• (a) preparing the sample for analysis, including adding a plurality of isotopically labeled peptides to the sample, each one of the isotopically labeled peptides producing, when ionized, a labeled peptide precursor ion of characteristic mass-to-charge (m/z) ratio;
• (b) chromotographically separating the sample;
• (c) ionizing the separated sample to generate sample ions;
• (d) performing an MS1 scan of the sample ions;
• (e) identifying a precursor ion in the MS1 scan that has a mass-to-charge ratio (m/z) matching an m/z of a labeled peptide precursor ion;
• (f) performing a first MS/MS analysis of the identified precursor ion to acquire a first MS/MS spectrum;
• (g) determining whether at least one peak is present in the first MS/MS spectrum matches one or more m/z's of a characteristic product ion or product ions generated by dissociation of the labeled peptide precursor ion;
• and
• (h) upon determination that the at least one peak is present in the first MS/MS spectrum, performing a second MS/MS analysis to measure an intensity of at least one product ion produced by dissociation of the endogenous peptide ion corresponding to the labeled precursor ion.