LC‐MS/MS isomeric profiling of permethylated N‐glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients

Abstract

Early stage detection and cancer treatment demand the identification of reliable biomarkers. Over the past decades, efforts have been devoted to assess the variation of glycosylation level as well as the glycan structures of proteins in blood or serum, associated with the development and/or progression of several cancers, including liver. Herein, an LC-MS/MS-based analysis was conducted to define the glycosylation patterns of haptoglobin glycoprotein derived from sera collected from cirrhotic and hepatocellular carcinoma (HCC) patients. The haptoglobin samples were extracted from serum using an antibody-immobilized column prior to the release of N-glycans. A comparison of non-isomeric and isomeric permethylated glycan forms was achieved using C18 and porous graphitic carbon (PGC) columns, respectively. In the case of C18-LC-MS/MS analysis, 25 glycan structures were identified of which 10 sialylated structures were found to be statistically significant between the two cohorts. Also, 8 out of 34 glycan structures identified by PGC-LC-MS/MS were found to be statistically significant, suggesting that isomeric distributions of a particular glycan composition were different in abundances between the two cohorts. The glycan isoform patterns distinguished early stage HCC from cirrhotic patients. Both retention times and tandem mass spectra were utilized to determine the specific isomeric glycan structures. All of the glycan isomers, which were statistically significant, were either branch fucosylated or composed of α-2,6 linked sialic acid moieties. The result of this study demonstrates the potential importance of isomeric separation for defining disease prompted aberrant glycan changes. The levels of several glycan isomers effectively distinguished early stage HCC from cirrhosis.

Glycoproteomic markers of hepatocellular carcinoma‐mass spectrometry based approaches

“…A different strategy developed by Mechref and coworkers (Huang et al, ) involved the use of isomeric profiling to identify glycan markers for cancer. Isomeric forms of glycans were separated and identified based upon separations with elevated temperatures in a PGC column followed by ESI‐MS in an Orbitrap Velos MS (see Fig.…”

Advances in mass spectrometry‐based glycomics

“…The capability of separating linkage isomers of sialic acid and galactose were demonstrated and confirmed via enzymatic digestion . Differences of isomer‐oriented profiles were observed in free oligosaccharides , N‐ and O‐ glycans in breast cancer cell lines and N‐ glycans released from blood serum haptoglobin of diseased patients . More details of LC separation and identifications of glycan isomers were reviewed in references .…”

LC–MS/MS glycomics of idiopathic rapid eye movement sleep behavior disorder

“…Student's t ‐tests were applied to absolute intensities acquired on C18‐LC–MS and PGC‐LC–MS to compare glycans and N‐glycan isomers between control and idiopathic RBD cohorts. Two‐tailed t ‐tests were performed, similar to what we have used in our previous studies using Markerview ™ software (AB Sciex, Framingham, MA) .…”

“…To address these issues, our group has adopted a sophisticated and advanced approach to optimize isomeric separation by using porous graphitized carbon (PGC) stationary phase under high temperature (75°C) , permitting reliable identification and characterization of glycan structures as well as precise and accurate quantitation of isomeric structures . Using this approach, in the present study, we performed comparative glycomic analyses in serum samples from nine patients with a clinical diagnosis of idiopathic RBD and ten healthy controls and obtained an accurate separation, identification, and quantification of isomeric glycans.…”