Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

Immunoglobulin (Ig)
glycosylation is recognized for its influence
on Ig turnover and effector functions. However, the large-scale profiling
of Ig glycosylation in a biomedical setting is challenged by the existence
of different Ig isotypes and subclasses, their varying serum concentrations,
and the presence of multiple glycosylation sites per Ig. Here, a high-throughput
nanoliquid chromatography (LC)- mass spectrometry (MS)-based method
for simultaneous analysis of IgG and IgA glycopeptides was developed
and applied on a serum sample set from 185 healthy donors. Sample
preparation from minute amounts of serum was performed in 96-well
plate format. Prior to trypsin digestion, IgG and IgA were enriched
simultaneously, followed by a one-step denaturation, reduction, and
alkylation. The obtained nanoLC-MS data were subjected to semiautomated,
targeted feature integration and quality control. The combined and
simplified protocol displayed high overall method repeatability, as
assessed using pooled plasma and serum standards. Taking all samples
together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were
attributable to 11 different peptide backbones, derived from IgG1,
IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA.
Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore,
previously reported associations of IgG Fc glycosylation with age
in healthy individuals were confirmed. In conclusion, the new method
paves the way for high-throughput multiprotein plasma glycoproteomics.