MassIVE MSV000093562

Partial Public PXD047513

Comparison of glycopeptide to released glycan abundances and the influence of glycopeptide charge state on N-linked glycosylation of IgG antibodies

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Description

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides with fluorescence abundances of released labeled glycans, both derived from therapeutic monoclonal antibodies. These proteins were selected for their very high purities, which assures the origin of all observed released glycans. 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. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high mannose and biantennary complex galactosylated and fucosylated and, in some cases, sialylated N-glycans. Except for Evolucumab, in-source ions derived from the loss of HexNAc or HexNAc and hexose sugars to HexNAc(4)Hex(4)Fuc(1) glycoform 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 across all mAb samples 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 closely to examine the effect of charge state on ion abundances. These results confirmed and refined results for the therapeutic proteins, clearly revealing a linear dependence of relative glycopeptide abundance on the mass of the glycan, with higher charge states favoring higher mass glycans. Overall, our 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. [doi:10.25345/C5VM4377R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: glycoproteomics ; glycomics ; mass spectrometry

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Principal Investigators:
(in alphabetical order) 		Concepcion Remoroza, NIST, USA
Submitting User: glycopep_2023
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