Oxygen isotopic substitution of peptidyl phosphates for modification-specific mass spectrometry

The first method of isotopic substitution of a nonbridging oxygen atom in pre-existing phosphates on peptides is reported, solving a long-standing, challenging issue in the sample preparation of phosphopeptides. Peptidyl phosphates, phosphate groups on phosphopeptides, are converted to phosphoramidates with carbodiimide assistance. Acid-catalyzed hydrolysis of the newly formed phosphoramidates incorporates one oxygen atom from H2(16)O or H2(18)O, producing peptidyl phosphates-16O1 or -18O1, respectively. The oxygen labels are stable under common separation and analysis conditions. This labeling method causes minimal structural alteration to peptidyl phosphates and allows the direct application of established phosphate-specific marker ions to the mass spectrometric analysis of differentially labeled phosphopeptide pairs. Using phosphotyrosinyl peptides as model analytes, the characteristic 16O1- and 18O1-labeled phosphotyrosine immonium ions at m/z 216.043 and 218.047 are used for developing a method of phosphopeptide quantitation that is independent of the amino acid sequence of the peptides. From analysis by tandem parallel fragmentation mass spectrometry, it is clear that the phosphate-specific marker ions authentically inherit the quantitative information from precursor phosphopeptides. The dynamic range for relative quantitation of differentially labeled phosphopeptides is at least 2 orders of magnitude for experiments run on a quadrupole time-of-flight mass spectrometer. The use of 16O1 and 18O1 labeling for counting the number of phosphate groups on peptides is also demonstrated.