Dataset Information

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

ABSTRACT: UNLABELLED:Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC-MS-MRM assay to quantify glycoforms of IgG subclasses 1-4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. An increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC-MS-MRM method for the quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC. BIOLOGICAL SIGNIFICANCE:We have demonstrated that both quantities and glycoforms of immunoglobulin subclasses change significantly in liver disease progression to HCC through quantitative study of immunoglobulin subclasses and their site specific glycoforms using a sensitive and selective LC-MS-MRM method. Redistribution of the glycoforms of specific immunoglobulin subclasses could have important implications for receptor mediated responses affecting the progression of liver disease.

SUBMITTER: Yuan W

PROVIDER: S-EPMC4329072 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

Yuan Wei W Sanda Miloslav M Wu Jing J Koomen John J Goldman Radoslav R

Journal of proteomics 20150109

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC-MS-MRM assay to quantify glycoforms of IgG subclasses 1-4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indic ...[more]

PMID: 25582524

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Project description:Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. BIOLOGICAL SIGNIFICANCE: We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease.

Project description:Epithelial ovarian cancer (EOC) was previously shown to be associated with glycosylation changes of total serum and total IgG proteins. However, as a majority of previous studies analyzed released glycan profiles, still little is known about IgG subclass-specific alterations in ovarian cancer. Hence, in this study, we investigated EOC-related glycosylation changes of the three most abundant IgG subclasses, namely, IgG1, IgG2 and IgG3 isolated from sera of 87 EOC patients and 74 age-matched healthy controls. In order to separate IgG2 and IgG3, we performed a two-step affinity purification employing Protein A and Protein G Sepharose. After tryptic digestion, IgG glycopeptides were enriched and measured by MALDI-TOF-MS. Finally, EOC-related glycosylation changes were monitored at the level of total agalactosylation, monogalactosylation, digalactosylation, sialylation, bisection and fucosylation, which were calculated separately for each IgG subclass. Interestingly, aside from an EOC-related increase in agalactosylation/decrease in monogalactosylation and digalactosylation observed in all IgG subclasses, some subclass-specific trends were detected. Glycosylation of IgG1 was found to be most strongly affected in EOC, as it exhibited the highest number of significant differences between healthy controls and EOC patients. Specifically, IgG1 was the only subclass that showed a significant decrease in sialylation and a significant increase in fucosylation in EOC patients. Interestingly, IgG2 and IgG3 that were often investigated collectively in previous studies, were found to have distinct glycosylation patterns. IgG3 displayed stronger EOC-related increase in agalactosylation/decrease in digalactosylation and was characterized by notably higher sialylation, which consequently decreased in EOC patients. In conclusion, our study indicates that IgG subclasses exhibit subtly distinct glycosylation patterns of EOC-related alterations and that IgG1 and IgG3 agalactosylation show the strongest association with CA125, the routine diagnostic marker. Additionally, our results show that simultaneous analyses of IgG2 and IgG3 might lead to wrong conclusions as these two subclasses exhibit noticeably different glycosylation phenotypes.

Dataset Information

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

Publications

Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease.

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