Tandem 18O stable isotope labeling for quantification of N-glycoproteome.

A new strategy using tandem 18O stable isotope labeling (TOSIL) to quantify the N-glycosylation site occupancy is reported. Three heavy oxygen atoms are introduced into N-glycosylated peptides: two 18O atoms are incorporated into the carboxyl terminal of all peptides during a tryptic digestion, and the third 18O atom is incorporated into the N-glycosylation site of asparagines-linked sugar chains specifically via a N-glycosidase F (PNGase F)-mediated hydrolysis. Comparing samples treated in H218O and samples treated in H216O, a unique mass shift of 6 Da can be shown for N-glycosylated peptide with single glycosylation site, which could be easily distinguished from those nonglycosite peptide pairs with a mass difference of 4 Da only. The relative quantities of N-glycosylated and its parent protein-levels were obtained simultaneous by measuring the intensity ratios of 18O/16O for glycosylated (6 Da) and for nonglycosylated (4 Da) peptides, respectively. Thus, a comparison of these two ratios can be utilized...