Glycans, Glycosite, and Intact Glycopeptide Analysis of N‑Linked Glycoproteins Using Liquid Handling Systems

Aberrant
glycosylation has been shown to associate with disease
progression, and with glycoproteins representing the major protein
component of biological fluids this makes them attractive targets
for disease monitoring. Leveraging glycoproteomic analysis via mass
spectrometry (MS) could provide the insight into the altered glycosylation
patterns that relate to disease progression. However, investigation
of large sample cohorts requires rapid, efficient, and highly reproducible
sample preparation. To address the limitation, we developed a high-throughput
method for characterizing glycans, glycosites, and intact glycopeptides
(IGPs) derived from N-linked glycoproteins. We combined disparate
peptide enrichment strategies (i.e., hydrophilic and hydrophobic)
and a liquid handling platform allowing for a high throughput and
rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip
workflow reduced sample processing time and facilitated the selective
enrichment of IGPs from complex samples. Furthermore, our approach
enabled the analysis of deglycosylated peptides and glycans from enriched
IGPs following PNGase F digest. Following development and optimization
of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin,
we investigated normal urine samples to obtain N-linked glycoprotein
information. Together, our method enables a high-throughput enrichment
of glycan, glycosites, and IGPs from biological samples.