GRable version 1.0: A software tool for site-specific glycoform analysis using the improved Glyco-RIDGE method with parallel clustering and MS2 information

Abstract

ABSTRACTHigh-throughput intact glycopeptide analysis is crucial to elucidate the physiological and pathological status of glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods have challenges due to the diversity and heterogeneity of glycan structures. Therefore, we have developed an MS1-based site-specific glycoform analysis method named “Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)” for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographical properties of glycopeptides and then searches each combination of core peptide and glycan composition by matching their mass and retention time differences. Here we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorisms, including “parallel clustering.” This unique function improved the comprehensiveness of glycopeptide detection and allowed analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the “confidence level” of the GRable results using MS2 information. This function facilitated reduced misassignment of core peptide and glycan composition and improved interpretation of the results. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large scale and in-depth. Therefore, this software will help to analyze the status and changes of glycans to obtain biological and clinical insights into protein glycosylations. GRable can run online freely using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).