Patent ID: 8227251

Claim:
A method of quantifying a peptide of interest in a sample comprising: (a) modifying the peptide of interest at terminus (1) with an isotopically heavy first tag (A*) and at terminus (2) with an isotopically light second tag (B) to form a structure of Formula (I): A*-Peptide-B (I); (b) adding an internal standard to the sample, wherein the internal standard comprises the sequence of the peptide of interest modified at terminus (1) with an isotopically light variant of the first tag (A) and at terminus (2) with an isotopically heavy variant of the second tag (B*), to form a structure of Formula (II): A-Peptide-B* (II), wherein the difference in mass between (A*) and (A) is equal to the difference in mass between (B*) and (B) such that the internal standard is isobaric to the modified peptide of interest; (c) obtaining a first mass spectrum of the sample; (d) identifying an ion in the first mass spectrum which corresponds to both the modified peptide of interest and the internal standard; (e) obtaining a second mass spectrum by collision-induced dissociation fragmentation of the ions of both the modified peptide of interest and the internal standard identified in step (d); (f) comparing the relative intensities of fragment ions of the modified peptide of interest and fragment ions of the internal standard; and (g) quantifying the peptide of interest based on the comparison from step (f).