Site-Specific Quantification of Surface N‑Glycoproteins in Statin-Treated Liver Cells

The
frequent modification of cell-surface proteins by N-linked
glycans is known to be correlated with many biological processes.
Aberrant glycosylation on surface proteins is associated with different
cellular statuses and disease progression. However, it is extraordinarily
challenging to comprehensively and site-specifically analyze glycoproteins
located only on the cell surface. Currently mass spectrometry (MS)-based
proteomics provides the possibility to analyze the N-glycoproteome,
but effective separation and enrichment methods are required for the
analysis of surface glycoproteins prior to MS measurement. The introduction
of bio-orthogonal groups into proteins accelerates research in the
robust visualization, identification, and quantification of proteins.
Here we have comprehensively evaluated different sugar analogs in
the analysis of cell-surface N-glycoproteins by combining copper-free
click chemistry and MS-based proteomics. Comparison of three sugar
analogs, N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine
(GlcNAz), and N-azidoacetylmannosamine (ManNAz), showed that metabolic
labeling with GalNAz resulted in the greatest number of glycoproteins
and glycosylation sites in biological duplicate experiments. GalNAz
was then employed for the quantification experiment in statin-treated
HepG2 liver cells, and 280 unique N-glycosylated sites were quantified
from 168 surface proteins. The quantification results demonstrated
that many glycosylation sites on surface proteins were down-regulated
in statin-treated cells compared to untreated cells because statin
prevents the synthesis of dolichol, which is essential for the formation
of dolichol-linked precursor oligosaccharides. Several glycosylation
sites in proteins that participate in the Alzheimer’s disease
pathway were down-regulated. This method can be extensively applied
for the global analysis of the cell-surface N-glycoproteome.