Fig. 4

Quantitative LC/MS/MS analysis of N-glycopeptides labeled by PNGase-mediated isotope-coded glycosylation site-specific differential labeling. Two N-glycopeptide samples are labeled differentially with PNGase in either \(H_2 ^{16} O\)(light: L) or \(H_2 ^{16} O\)(heavy: H), combined, and analyzed by LC/MS/MS. The molecular mass of a deglycosylated peptide increases by 1Da (L) or 3Da (H) unit from the calculated mass by the PNGase mediated conversion of N-glycosylated Asn to Asp. The relative content of peptide in the two N-glycopeptide samples is estimated from the signal ratio between the “light” and “heavy” peptides, after correction of the signal overlaps because of natural abundance of isotopes. a Mass spectrum of a deglycosylated peptide of human transferrin, residues 421–433: CGLVPVLAENYNK, treated in \(H_2 ^{18} O\). b, c Mass spectra of a mixture of peptides treated with \(H_2 ^{16} O\)(L) and \(H_2 ^{18} O\)(H) at a ratio of 1:3 and 1:1, respectively