Urinary Glycopeptide Analysis for the Investigation of Novel Biomarkers

PURPOSE: Urine is a rich source of potential biomarkers, including glycoproteins. Glycoproteomic analysis remains difficult due to the high heterogeneity of glycans. Nevertheless, recent advances in glycoproteomics software solutions facilitate glycopeptide identification and characterization. The aim is to investigate intact glycopeptides in the urinary peptide profiles of normal subjects using a novel PTM-centric software-Byonic. EXPERIMENTAL DESIGN: The urinary peptide profiles of 238 normal subjects, previously analyzed using CE-MS and CE-MS/MS and/or LC-MS/MS, are subjected to glycopeptide analysis. Additionally, glycopeptide distribution is assessed in a set of 969 patients with five different cancer types: bladder, prostate and pancreatic cancer, cholangiocarcinoma, and renal cell carcinoma. RESULTS: A total of 37 intact O-glycopeptides and 23 intact N-glycopeptides are identified in the urinary profiles of 238 normal subjects. Among the most commonly identified O-glycoproteins are Apolipoprotein C-III and insulin-like growth factor II, while titin among the N-glycoproteins. Further statistical analysis reveals that three O-glycopeptides and five N-glycopeptides differed significantly in their abundance among the different cancer types, comparing to normal subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Through the established glycoproteomics workflow, intact O- and N-glycopeptides in human urine are identified and characterized, providing novel insights for further exploration of the glycoproteome with respect to specific diseases.