Sweet-Heart - An integrated suite of enabling computational tools for automated MS2/MS3 sequencing and identification of glycopeptides

High efficiency identification of intact glycopeptides from a shotgun glycoproteomic LC-MS2 dataset remains problematic. The prevalent mode of identifying the de-N-glycosylated peptides is littered with false positives and addresses only the issue of site occupancy. Here, we present Sweet-Heart, a computational tool set developed to tackle the heart of the problems in MS' sequencing of glycopeptide. It accepts low resolution and low accuracy ion trap MS2 data, filters for glycopeptides, couples knowledge-based de novo interpretation of glycosylation-dependent fragmentation pattern with protein database search, and uses machine-learning algorithm to score the computed glyco and peptide combinations. Higher ranking candidates are then compiled into a list of MS2/MS3 entries to drive subsequent rounds of targeted MS3 sequencing of putative peptide backbone, allowing its validation by database search in a fully automated fashion. With additional fishing out of all related glycoforms and final data integration, the platform proves to be sufficiently sensitive and selective, conducive to novel glycosylation discovery, and robust enough to discriminate, among others, N-glycolyl neuraminic acid/fucose from N-acetyl neuraminic acid/hexose. A critical appraisal of its computing performance shows that Sweet-Heart allows high sensitivity comprehensive mapping of site-specific glycosylation for isolated glycoproteins and facilitates analysis of glycoproteomic data. Biological significance The biological relevance of protein site-specific glycosylation cannot be meaningfully addressed without first defining its pattern by direct analysis of glycopeptides. Sweet-Heart is a novel suite of computational tools allowing for automated analysis of mass spectrometry-based glycopeptide sequencing data. It is developed to accept ion trap MS2/MS3 data and uses a machine learning algorithm to score and rank the candidate peptide core and glycosyl substituent combinations. By eliminating the need for manual, labor-intensive, and subjective data interpretation, it facilitates high throughput shotgun glycoproteomic data analysis and is conducive to identification of unanticipated glycosylation, as demonstrated here with a recombinant EGFR. (C) 2013 Elsevier B.V. All rights reserved.