Quantifying Peptides in Isotopically Labeled Protease Digests by Ion Mobility/Time-of-Flight Mass Spectrometry

Ion mobility/time-of-flight techniques have been used to analyze mixtures of isotopically labeled peptides. The isotopic labels were generated by treatment of peptides with N-acetoxysuccinimide (or the deuterated analogue), which results in acetylation (or deuterioacetylation) of the primary amines (i.e., the N-terminus and lysine residues). The relative concentrations of a peptide in each sample are determined by comparing the peak intensities for isotopic pairs. An important consideration is that as mixtures become increasingly complex, isotopic pairs of peaks may overlap with other peaks in the mass spectrum. The influence of the acetyl and deuterioacetyl groups on the mobilities of peptides is considered. The coincidence in mobilities of isotopic pairs provides a means of distinguishing isotopic pairs from other isobaric interferences.