Method for highly multiplexed quantitation of peptides by mass spectrometry and mass labels therefor

A method of measuring the relative abundances of peptide analytes by mass spectrometry, comprising:

• preparing a plurality of peptide-containing samples;
• reacting each sample with an isobaric labeling reagent selected from an isobaric labeling reagent set to create differentially labeled peptides, the isobaric labeling reagent set consisting of a plurality of different isobaric labeling reagents wherein:
• each isobaric labeling reagent comprises a reporter group, a mass normalizing group, a peptide-reactive group, and a cleavable linker attaching the reporter group to the mass normalizing group, each one of the plurality of isobaric labeling reagents having the same nominal mass;
• each one of the isobaric labeling reagents has a reporter group with a mass different from the masses of the reporter groups of each of the other isobaric labeling reagents;
• and
• a first collection of at least two of the isobaric labeling reagents have reporter groups of the same nominal mass but different exact mass by having different numbers of at least one of 13C, 15N and 2H substitutions;
• wherein each sample is reacted with a unique one of the isobaric labeling reagents, such that labeled peptides in each sample have a reporter group mass that is different from the reporter group mass of the labeled peptides in each of the other samples, and wherein at least two of the samples are reacted with corresponding isobaric labeling reagents of the first collection;
• combining the samples;
• ionizing the combined samples to generate labeled peptide ions;
• fragmenting the labeled peptide ions to produce reporter ions, the reporter ions having a mass-to-charge ratio uniquely characterizing one of the samples;
• and
• measuring the abundances of the reporter ions at each characteristic mass-to-charge ratio using a mass analyzer.