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.

N连接聚糖对细胞表面蛋白的频繁修饰，已知与诸多生物学过程密切相关。细胞表面蛋白的糖基化异常，与不同细胞状态及疾病进展紧密相关。然而，仅针对细胞表面糖蛋白开展全面且位点特异性的分析仍极具挑战。当前基于质谱（mass spectrometry, MS）的蛋白质组学技术为N糖蛋白组的分析提供了可能，但在质谱检测前，需借助有效的分离富集手段以靶向分析表面糖蛋白。将生物正交基团引入蛋白体系，可推动蛋白的高效可视化、鉴定与定量研究。本研究结合无铜点击化学与基于质谱的蛋白质组学技术，系统评估了多种糖类似物在细胞表面N糖蛋白组分析中的应用效果。通过对比N-叠氮乙酰半乳糖胺（N-azidoacetylgalactosamine, GalNAz）、N-叠氮乙酰葡糖胺（N-azidoacetylglucosamine, GlcNAz）及N-叠氮乙酰甘露糖胺（N-azidoacetylmannosamine, ManNAz）三种糖类似物，研究发现采用GalNAz进行代谢标记时，生物学重复实验中可获得最多的糖蛋白及糖基化位点数量。随后，本研究选用GalNAz开展了他汀处理的HepG2肝癌细胞的定量实验，从168种表面蛋白中共定量到280个独特的N糖基化位点。定量结果显示，相较于未处理组，他汀处理细胞表面蛋白的诸多糖基化位点呈现下调趋势——这是由于他汀会抑制多萜醇的合成，而多萜醇是形成多萜醇连接的前体寡糖所必需的物质。参与阿尔茨海默病通路的多种蛋白的糖基化位点亦出现下调。本方法可广泛应用于细胞表面N糖蛋白组的全景分析。