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.

Systematic investigation of cellular response and pleiotropic effects in atorvastatin-treated liver cells by MS-based proteomics.

For decades, statins have been widely used to lower cholesterol levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is well-known that statins have pleiotropic effects including improving endothelial function and inhibiting vascular inflammation and oxidation. However, the cellular responses to statins and corresponding pleiotropic effects are largely unknown at the proteome level. Emerging mass spectrometry-based proteomics provides a unique opportunity to systemically investigate protein and phosphoprotein abundance changes as a result of statin treatment. Many lipid-related protein abundances were increased in HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE, and LDLR, while the abundances of proteins involved in cellular response to stress and apoptosis were decreased. Comprehensive analysis of protein phosphorylation demonstrated that several basic motifs were enriched among down-regulated phosphorylation sites, which indicates that kinases with preference for these motifs, such as protein kinase A and protein kinase C, have attenuated activities. Phosphopeptides on a group of G-protein modulators were up-regulated, which strongly suggests that cell signal rewiring was a result of the effect of protein lipidation by the statin. This work provides a global view of liver cell responses to atorvastatin at the proteome and phosphoproteome levels, which provides insight into the pleiotropic effects of statins.