Comparative proteomic analysis of de‐N‐glycosylated serum from hepatitis B carriers reveals polypeptides that correlate with disease status

Abstract

Analysis of the polypeptide profile in tissues, cells, and sera by high-resolution two-dimensional (2-D) gel electrophoresis offers promise in the identification of biomarkers that correlate with disease. However, sera contain many polypeptides bearing N-linked glycosylation that can complicate interpretation. Therefore, we tested the possibility that de-N-glycosylation of the polypeptides present in human serum would result in a simplification of serum proteome profiles. Briefly, polypeptides present in human serum were left untreated or subjected to de-N-glycosylation by incubation with PNGase F and resolved by high-resolution 2-D gel electrophoresis. De-N-glycosylation reduced the number of glycoform variants, enhanced the resolution of many polypeptides and allowed other polypeptides to become visible. As an initial test of concept, clinically relevant serum samples from individuals with or without diagnosis of hepatocellular carcinoma were compared. Several polypeptides, apparent only after de-N-glycosylation, were shown to correlate with disease. Although the results are preliminary and the identities of all the putative biomarkers not yet known, the data suggest that de-N-glycosylation offers a method to enhance the resolution of serum polypeptide profiles and has value in comparative proteomic studies.

SELDI-TOF MS profiling of serum for detection of the progression of chronic hepatitis C to hepatocellular carcinoma

“…[19][20][21]38 To date, new HCC biomarkers have been sought primarily by using differential two-dimensional gel separations of tumor tissues 38 or serum 19,21 together with mass spectrometry for protein identification. This is a productive discovery approach for identifying biomarker proteins of masses greater than 10 kd; however, the low sample throughput limits the development of direct diagnostic assays.…”

“…18 Proteomic techniques are increasingly being applied to identify biomarkers in serum indicative of advanced liver disease, particularly hepatitis and HCC. [19][20][21] One of the recent technological advances in proteomics is the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). [22][23][24] Applications of this technology to clinical fluids have suggested great potential for the early detection of prostate, breast, headneck, ovarian, pancreatic, bladder, and liver cancers.…”

Human body fluid proteome analysis

“…Deglycosylation of serum proteins may result in a simplification of serum proteome profiles and enhanced resolution for comparative proteome analysis. Serum amyloid P component and ceruloplasmin, apparent only after de-N-glycosylation, were shown to correlate with HBV disease [247]. Golgi Protein 73 (GP73), a glycoprotein, was found elevated and hyperfucosylated in sera of patients with hepatocellular carcinoma, which may assist in the early detection of HBV-induced liver cancer [155].…”

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

“…Nglycosylation can influence proteomic analysis of such samples, as a key biological role of Nglycosylation is to protect glycoproteins from proteolytic cleavage, an effect still present following protein denaturation. It has been reported that removal of N-glycans by glycosidases such as PNGase F can improve MS identification of N-glycoproteins [23][24][25][26]. However, a deglycosylation step is not generally included in proteomic workflows.…”