Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease

Abstract

Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

“…Formations of Lewis type epitopes such as SLeX, LeX, or LeY were recently observed on liver secreted glycoproteins [17–21], in addition to the well-known increase in core fucosylated α- fetoprotein in hepatocellular carcinoma [22–24]. These alterations in the glycosylation of proteins were extensively studied in human tissues and especially in serum [13,15,25–27]. Other described alterations of glycans include increased branching, sialylation, agalactosylated glycans or polylactosamine chains [16,28,29].…”

“…We and others have recently shown that this workflow can be used for quantification of glycopeptides [13,25, 43–46]. Collision induced dissociation of glycopeptides (CID) yields high intensity oxonium ions that originate from the glycans and less intense peptide-glycan or peptide fragments.…”

“…Collision induced dissociation of glycopeptides (CID) yields high intensity oxonium ions that originate from the glycans and less intense peptide-glycan or peptide fragments. The highly sensitive but less specific oxonium ions are typically used as quantitative transitions in the MRM mode [13,25,47]. In complex sample mixture, the most intense peptide-glycan fragments are often selected as specific transitions used for glycopeptide confirmation.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Several applications of multiple reaction monitoring (MRM)-based glycopeptide analysis from a variety of biological systems and diseases were lately published including the use of MRM-based serum glycoproteomics to study esophagus diseases (140), liver disease (141), and the immunoglobulin subclasses (134,142,143). Among other technical challenges unique to MRM-based glycopeptide detection and quantitation, one of the most important considerations is the selection of the precursor-product transitions being monitored.…”

A case for protein-level and site-level specificity in glycoproteomic studies of disease

“…For instance, Benicky et al evaluated the site-specific core fucosylation for hemopexin (HPX; a glycoprotein with five N-glycosylation sites) and complement factor H (CFH; a glycoprotein with nine Nglycosylation sites) in patients with liver cirrhosis and hepatocellular carcinoma (HCC) [57]. HPX and CFH were isolated from blood samples and subjected to trypsin digestion, followed by digestion with neuraminidase and then endoglycosidase F2 and F3 (Endo F2 and F3).…”