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

Abstract

BackgroundNon-invasive monitoring of liver disease remains an important health issue. Liver secreted glycoproteins reflect pathophysiological states of the organ and represent a rational target for serologic monitoring. In this study, we describe sialylated O-glycoforms of liver-secreted hemopexin (HPX) and quantify them as a ratio of disialylated to monosialylated form (S-HPX).MethodsWe measured S-HPX in serum of participants of the HALT-C trial using a LC–MS/MS-MRM assay.ResultsRepeated measurements of S-HPX in the samples of 23 disease-free controls, collected at four different time points, show that the ratio remains stable in the healthy controls but increases with the progression of liver disease. The results of measurement of S-HPX in serum of participants of the HALT-C trial show that it increased significantly (Kruskal–Wallis test, p < 0.01) in liver disease as the stage of fibrosis progressed in liver biopsies. We observed a 1.7-fold increase in fibrosis defined as Ishak score 3–4 (24.9 + 14.2, n = 22) and 4.7-fold increase in cirrhosis defined as Ishak score 5–6 (68.6 + 38.5; n = 24) compared to disease-free controls (14.7 + 6.7, n = 23). S-HPX is correlated with AFP, bilirubin, INR, ALT, and AST while inversely correlated with platelet count and albumin. In an independent verification set of samples, S-HPX separated the Ishak 5–6 (n = 15) from the Ishak 3–4 (n = 15) participants with AuROC 0.84; at the same time, the Ishak 3–4 group was separated from disease-free controls (n = 15) with AuROC 0.82.ConclusionS-HPX, a measure of sialylated O-glycoforms of hemopexin, progressively increases in fibrotic and cirrhotic patient of HCV etiology and can be quantified by an LC–MS/MS-MRM assay in unfractionated serum of patients. Quantification of sialylated O-glycoforms of this liver secreted glycoprotein represents a novel measure of the stage of liver disease that could have a role in monitoring the progression of liver pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9125-x) contains supplementary material, which is available to authorized users.

N and O glycosylation of the SARS-CoV-2 spike protein

“…GluC, PNGase F, neuraminidase and beta galactosidase digests of tryptic peptides were carried out as described previously (11,12) with heat inactivation (99 °C for 10 min) prior to the addition of any enzyme.…”

“…We have used the m/z 657 ion to assign sialylation of the core-2 monosialylated structures. We used an optimized procedure based on a hemopexin glycopeptide standard, which we described previously (12), and we determined CCS of the fragment 657 ( Figure 6) observed by fragmentation of the glycopeptide with sialyl-T antigen with linkage (α2-3) (CCS 234.9) and by fragmentation of an Nglycopeptide with sialyl-LacNAc with (α2-6) linkage (CCS 232.8) (data not shown). This is in agreement with the previously published results on the linkages of the sialylated glycans (6,8).…”

Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM

“…Serum samples of 15 disease-free controls were collected at Georgetown University Medical Center under protocols approved by the Institutional Review Board. De-identified serum samples of 30 patients with various degree of liver fibrosis were obtained from the hepatitis C antiviral long-term treatment against cirrhosis trial (HALT-C), as described in our recent study [35]. The HALT-C trial, a prospective randomized controlled trial of 1050 patients, evaluated effect of long-term low-dose peginterferon alpha-2a (IFN) in participants that failed initial anti-HCV therapy with interferon [37,38].…”

“…Liver disease status of the HALT-C participants was classified into fibrosis (Ishak score 3–4) or cirrhosis (Ishak score 5–6) based on biopsy-evaluation. All the participants selected for this study were from the control arm of the HALTC trial that did not receive IFN treatment [35]. Method optimization was carried out on a sample pooled from the 45 serum samples of all participants (equal volume of each) and the pooled sample served as a quality control (QC) that was analyzed in parallel with the analyses of the 45 study samples.…”

A Rapid LC-MS/MS-PRM Assay for Serologic Quantification of Sialylated O-HPX Glycoforms in Patients with Liver Fibrosis

“…In this study, we developed a mLC-MS/MS-PRM assay for the quantification of site-specific mucin-type O-glycoforms of hemopexin, which we previously reported as a promising candidate biomarker for the serologic monitoring of liver fibrosis [ 11 , 12 ]. We have shown that the sialylated O-glycoforms of hemopexin (HPX) in serum of patients are associated with advancing fibrosis in hepatitis C-associated liver disease [ 11 ].…”