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
posted on 2024-03-07, 12:22authored byConcepcion A. Remoroza, Meghan C. Burke, Tytus D. Mak, Sergey L. Sheetlin, Yuri A. Mirokhin, Brian T. Cooper, Zachary C. Goecker, Mark S. Lowenthal, Xiaoyu Yang, Guanghui Wang, Dmitrii V. Tchekhovskoi, Stephen E. Stein
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).