Increased sialylation of site specific O-glycoforms of hemopexin in liver disease

Additional file 1 . Supplemental methods, figures and tables. SM 1.1. Mapping of the O-glycosylation sites. SM 1.2. Beta elimination and mass spectrometric analysis of HPX O-glycans. Table S1. Basic characteristics of disease-free controls and HALT-C participants. Table S2. The impact of IFN treatment on S-HPX. Groups of fibrotic and cirrhotic participants in the HALT-C trial were separated into the IFN treated and control arms. Table S3. Model estimates for logistic regression model in discovery set. Table S4. S-HPX measurement in the discovery and validation sets of participants. Figure S1. Precursor mass spectra confirmation of complete desialylation of HPX using 2M Acetic acid. Figure S2. ETD spectra of sialidase-treated O-glycopeptides corresponding to HILIC fractions of mono- (top), bis- (middle), and triply-glycosylated (bottom) O-glycopeptide of HPX. Figure S3. Direct quantification of S-HPX at progressing stages of liver disease divided by gender (left) and race (right; CA Caucasian, AA African-American). Figure S4. Significant associations of S-HPX and other clinical variables. Figure S5. Quantification of detected N-glycopeptides at three different N-glycosylation sequons (N64, N187 and N453) of HPX

A multiplexed microflow LC–MS/MS-PRM assay for serologic quantification of IgG N- and HPX O- glycoforms in liver fibrosis

Abstract Targeted quantification of glycoproteins has not reached its full potential because of limitations of the existing analytical workflows. In this study, we introduce a targeted microflow LC–MS/MS-PRM method for the quantification of multiple glycopeptides in unfractionated serum samples. The entire preparation of 16 samples in a batch is completed within 3 h, and the LC–MS quantification of all the glycoforms in a sample is completed in 15 min in triplicate, including online capture and desalting. We demonstrate applicability of the workflow on a multiplexed quantification of eight N-glycoforms of immunoglobulin G (IgG) together with two O-glycoforms of hemopexin (HPX). We applied the assay to a serologic study of fibrotic liver disease in patients of HCV etiology. The results document that specific IgG- and HPX-glycoforms detect efficiently fibrotic disease of different degree, and suggest that the LC–MS/MS-PRM assays may provide rapid and reproducible biomarker assay targeting simultaneously the N- and O-glycoforms of the peptides. We propose that such high throughput multiplexed methods may advance the clinical use of the LC–MS/MS assays

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

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

Protein and Site Specificity of Fucosylation in Liver-Secreted Glycoproteins

Chronic liver diseases are a serious health problem worldwide. One of the frequently reported glycan alterations in liver disease is aberrant fucosylation, which was suggested as a marker for noninvasive serologic monitoring. We present a case study that compares site specific glycoforms of four proteins including haptoglobin, complement factor H, kininogen-1, and hemopexin isolated from the same patient. Our exoglycosidase-assisted LC–MS/MS analysis confirms the high degree of fucosylation of some of the proteins but shows that microheterogeneity is protein- and site-specific. MSn analysis of permethylated detached glycans confirms the presence of LeY glycoforms on haptoglobin, which cannot be detected in hemopexin or complement factor H; all three proteins carry Lewis and H epitopes. Core fucosylation is detectable in only trace amounts in haptoglobin but with confidence on hemopexin and complement factor H, where core fucosylation of the bi-antennary glycans on select glycopeptides reaches 15–20% intensity. These protein-specific differences in fucosylation, observed in proteins isolated from the same patient source, suggest that factors other than up-regulation of enzymatic activity regulate the microheterogeneity of glycoforms. This has implications for selection of candidate proteins for disease monitoring and suggests that site-specific glycoforms have structural determinants, which could lead to functional consequences for specific subsets of proteins or their domains

Extracellular Heparan 6-<i>O</i>-Endosulfatases SULF1 and SULF2 in Head and Neck Squamous Cell Carcinoma and Other Malignancies

Pan-cancer analysis of TCGA and CPTAC (proteomics) data shows that SULF1 and SULF2 are oncogenic in a number of human malignancies and associated with poor survival outcomes. Our studies document a consistent upregulation of SULF1 and SULF2 in HNSC which is associated with poor survival outcomes. These heparan sulfate editing enzymes were considered largely functional redundant but single-cell RNAseq (scRNAseq) shows that SULF1 is secreted by cancer-associated fibroblasts in contrast to the SULF2 derived from tumor cells. Our RNAScope and patient-derived xenograft (PDX) analysis of the HNSC tissues fully confirm the stromal source of SULF1 and explain the uniform impact of this enzyme on the biology of multiple malignancies. In summary, SULF2 expression increases in multiple malignancies but less consistently than SULF1, which uniformly increases in the tumor tissues and negatively impacts survival in several types of cancer even though its expression in cancer cells is low. This paradigm is common to multiple malignancies and suggests a potential for diagnostic and therapeutic targeting of the heparan sulfatases in cancer diseases