High Mannose and Hybrid Glycosylation of Human Serum Proteins in Liver Disease

摘要

Our study is focused on the site specific analysis of oligomannose and hybrid glycans,. These incompletely processed glycoforms are secreted by the liver and other tissues together with the more common complex glycoforms. And we have observed increase of these glycoforms in liver disease by analysis of detached N-glycans. Here we focus on the analysis of the site-specific glycoforms associated with specific proteins. Glycosidase-assisted HR mass spectrometry combined with GPS software analysis allowed us to identify a set of high mannose and hybrid glycopeptides in the serum glycoproteome of patients with liver disease (cirrhosis or hepatocellular carcinoma). Tryptic digests of glycoproteins were analyzed by HR LC-MS/MS using 5600 tripleTOF mass spectrometer working in DDA mode following ConA lectin enrichment of oligomannose and hybrid glycopeptides. Glycopeptides were separated on C18 capillary column using 90 min acetonitrile gradient. PNGase F digestion was used for preparation of de-glycosylated peptides by chromatography identical to the chromatography of the glycopeptides. Data were processed using Protein pilot software and the identified peptides were used for glycopeptide analysis using the GPS software. All glycopeptide spectra were confirmed manually. We were able to identify more than fifty high mannose and hybrid glycopeptides in human serum using combination of selective structure enrichment, chromatographic reproducibility, HR mass spectrometric analysis, glycopeptide database, and GPS software. Immunoaffinity depletion of the most abundant serum proteins revealed additional glycopeptides which were masked in crude serum by oligomannosylated peptides derived from abundant glycoproteins such as immunoglobulins or complement proteins. These uncovered oligomannose and hybrid glycopeptides originate from liver secreted glycoproteins and low abundant immune cell proteins which represent a minor fraction of site specific glycoforms in healthy subjects. In summary, we identified a number of high mannose and hybrid glycopeptides associated with liver disease progression by the combination of label free mass spectrometric quantification of protein and simultaneous site specific quantification of glycopeptides.