Comprehensive glycomic analysis of haptoglobin in the serum of patients with gastric cancer

Abstract

Gastric cancer has one of the highest cancer mortality rates worldwide, in large part owing to difficulties related to the early-stage detection of this type of cancer. We developed a comprehensive glycomic approach involving an immunoaffinity analysis, lectin blotting, and advanced MS techniques with regard to the discovery of potential gastric cancer biomarkers. Lectin blotting was initially used to identify a target glycoprotein that displayed aberrant glycosylation during cancer development. Of the major serum glycoproteins, haptoglobin which is identified by in-gel digestion and an MS analysis showed the most notable changes in lectin-binding affinity and was therefore selected as a target for subsequent analysis.

We purified haptoglobin from 16 serum samples consisting of gastric cancer patients (n=10) and control subjects (n=6) by immunoaffinity. N-glycans were directly released from haptoglobin using PNGaseF and were analyzed by MALDI-MS and LC-MS to obtain quantitative and structural information. An ANOVA statistical analysis was conducted to identify potential biomarkers for gastric cancer cases. In general, increased levels of antennary branching with fucosyl residues, and decreased levels of high mannose-type structures were observed in gastric cancer patients compared with controls. Fucosylated complex-type glycan Hex6HexNAc5Fuc1, 6510, and fucosylated complex-type glycan Hex6HexNAc5Fuc1NeuAc1, 6511, of Hp were found as fucosylated biomarker candidates with high AUC values (0.97, 0.98) between normal and cancer patients. A structural analysis by LC/MS/MS showed that the fucosylated N-glycans from patient haptoglobin displayed primarily antennary fucosylation, which can be categorized as Lex or sialyl-$Le^x$ type structures.

To analyze haptoglobin glycosylation in a site-specific manner, a targeted glycoproteomics approach was developed and applied to serum from gastric cancer patients. Haptoglobin was subjected to glyco-analytical multispecific proteolysis (glyco-AMP) in order to generate site-specific glycopeptides. Glycopeptides were identified and quantified by structure-specific porous graphitized carbon nano-LC/MS and -LC/MS/MS. From haptoglobin’s four glycosylation sites, a total of 96 glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked and quantified across all cancer and control samples. Differences in abundance levels between cancer and control samples were marked by particularly high magnitudes, e.g., fucosylated complex-type glycans at Asn-241 increased in abundance by nearly ten-fold during gastric cancer. Glycan/glycosite pairings were screened for their suitability as potential biomarkers. Two glycan/glycosite pairings (Hex7HexNAc6Fuc1 at Asn-241 and Hex6HexNAc5Fuc1NeuAc1 at Asn-241) exhibited exceptionally high control-to-cancer fold changes along with ROC curve areas of 1.0, indicating perfect discrimination between the controls and gastric cancer cases (including both early- and late-stage patients). This comprehensive glycomic and targeted glycoproteomics approach using immunoaffinity, lectin blotting, and an advanced MS analysis may facilitate effective gastric cancer screening for the early diagnosis of gastric cancer.