Liquid Chromatography Mass Spectrometry-based O-glycomics To Evaluate Glycosylation Alterations In Gastric Cancer

Objective:To detect the expression of O-glycans and find out the differences in serum of gastric cancer patients and healthy controls by glycomics method. To establish the database of O-glycans, and identify the aberrant O-glycan structures specific to gastric cancer, and to evaluate its value in diagnosing gastric cancer. Our works for glycomics, the analysis about qualitative and quantitative of O-glycans, may provide a basis for research of other serum samples.Methods:1) We investigated the differences in O-glycosylation in the serum of 157 gastric cancer patients (GC) and 144 healthy donors, using the method of rapid one-pot nonreductive O-glycans released from glycoproteins and labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP); 2) Released and labeled O-glycans were purified by Carbograph graphitized carbon SPE cartridges; 3) PMP-labeled O-glycans were subjected to qualitative and quantitative analysis in the positive mode on the High-Performance LC Mass Spectrometer; 4) EIC peaks were extracted from corresponding m/z ions after MS profiling of each experimental sample collected by Xcalibur; 5) All EIC peaks were analyzed by multivariate statistics, student t tests and receiver operating characteristic (ROC); 6) Correlation analyses for the differential O-glycans and clinical tumor biomarkers; 7) The expression of MUC1 and Tn-MUCl in gastric cancer tissues were analyzed by immunohistochemical.Results:1) The expression level of TF antigen (corel), core2, ST antigen, and core2 complex O-glycans (m/z 733.33, m/z 809.42) were increased significantly (P< 0.0001) in the serum of gastric cancer patients; 2) Core3, m/z 529.75, and diST -antigen were decreased in the serum of gastric cancer patients compared with controls (P< 0.001); 3) The structural information of above O-glycans were clear by LC-MS/MS analysis; 4) In addition, according to Pearson correlation analysis, there were significant correlations between the abundance of the O-glycans and glycoproteins (MUC1, CEA) in the serum of GC; 5) Moreover, by immunohistochemical analysis, MUC1 was upregulated and aberrantly glycosylated in gastric cancer tissues versus para-carcinoma tissues.Conclusion:In our study, we have achieved the qualitative and quantitative analysis of O-glycans according to one-pot nonreductive O-glycans released from glycoproteins and labeled with 1-phenyl-3-methyl-5-pyrazolone, followed by LC-MS and LC-MS/MS analysis. We have confirmed that the differences in O-glycosylation in the serum of 157 gastric cancer patients (GC) and 144 healthy donors. There were significant correlations between the abundance of the O-glycans and glycoproteins (MUC1, CEA) in the serum of GC. The O-glycosylation was aberrant in gastric cancer tissues.