Comparison of N-Glycopeptide to Released N-Glycan Abundances and the Influence of Glycopeptide Mass and Charge States on N-Linked Glycosylation of IgG Antibodies

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).