Discovery Of Site-specific Glycan Biomarkers For Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma(ICC)is a primary malignancy which originates from the secondary bile ducts and above the peripheral intrahepatic bile ducts,and the incidence and mortality rate have shown an increasing trend year by year in recent years.Patients with ICC have no characteristic symptoms in the early stage,routine screening methods are difficult to diagnose.Most of them cannot be distinguished from hepatocellular carcinoma(HCC)by imaging,and when they are finally diagnosed already at an advanced stage,missing the optimal period of treatment and significantly reducing their survival rate.Therefore,the early and accurate diagnosis of ICC is of great significance.Serum is one of the ideal clinical diagnostic samples due to its ease of sampling and high sensitivity.Serum marker Carbohydrate Antigen19-9(CA19-9)is currently used as diagnostic and prognostic indicator for ICC,but its expression is elevated in only about 50% of ICC.Studies have shown that abnormal glycosylation is associated with the development of cancer.With the development of mass spectrometry technology,glycoproteomics will bring hope for screening for tumor markers that are detected and monitored early in cancer.Therefore,finding specific and sensitive ICC tumor markers that can be distinguished from HCC will help to diagnose and treat ICC patients in a timely and accurate manner.In this study,we first performed N-glycoproteomics analysis based on intact Nglycopeptides and TMT labeling quantification on serum samples from 7 ICC patients,6HCC patients and 6 healthy volunteers.A total of 1478 intact N-glycopeptides were identified,containing 85 N-glycan compositions corresponding to 173 sugar chain structures.By analyzing glycan type,it was found that most of the serums in these 19 cases were complex sugar chains,and more than half of the sugar chains had sialic acid modifications.By quantitatively comparing the intact N-glycopeptide changes in ICC and HCC serum by TMT labeling method,74 differential intact N-glycopeptides based on sugar chain composition were screened for specific changes in ICC and HCC,respectively.GO and KEGG analysis revealed that the glycoproteins corresponding to these differential glycopeptides are mainly involved in biological processes such as the complement and coagulation cascades and activation protein cascades.Since a sugar chain composition corresponds to multiple isomers,we further quantified intact N-glycopeptide according to the sugar chain structure.By integrating differential N-glycopeptides based on the screening of sugar chain composition and sugar chain structure,5 important glycoproteins were finally screened,and the changes in glycosylation modifications at different sites of these glycoproteins in ICC and HCC were significantly different,and had the potential as biomarkers.To verify the accuracy of potential intact glycopeptide biomarkers obtained by TMT labeling quantitative screening,we analyzed;intact N-glycopeptides in serum from another group of ICC patients,HCC patients and healthy volunteers(6 each)by Label-free quantitative omics.By comparing the intact glycopeptides quantification results of the discovery cohort and the validation cohort based on the sugar chain composition and sugar chain structure,we finally obtained three glycopeptides candidate biomarkers at different glycosylation sites on haptoglobin(HP),which were further analyzed by the Receiver Operating Characteristic curve.ROC curve analysis showed that the area under the curve was above 0.7.These ICCspecific intact glycopeptides that can be distinguished from HCC are expected to serve as potential serum markers for ICC.In summary,this study screened intact N-glycopeptide with ICC specific changes by TMTlabeled quantitative omics analysis of intact N-glycopeptide in the serums of ICC patients,HCC patients and healthy volunteers.The potential serum markers for ICC were then further identified by validation in new serum samples by Label-free quantitative methods,which will provide strong data support for timely and accurate diagnosis and prognosis of ICC.