GlycoMiner: a new software tool to elucidate glycopeptide composition

Abstract

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.

Fragmentation characteristics of glycopeptides

“…This has high relevance for structure analysis, as this ion clearly indicates the mass of the peptide chain (and therefore can be used to identify which glycoprotein gives rise to the studied glycopeptide) [13,20]. This ion may fragment further loosing the last GlcNAc unit, yielding a singly protonated peptide ion ( [Pep + H] + ).…”

N- and O-Glycosylation in the Murine Synaptosome

“…Software exists that can address some of these issues in the case of N-glycosylation (28,29). Site assignment for Nlinked glycopeptides is aided by the fact that these peptides typically will have a single asparagine residue that matches the NXS/T/C motif.…”

A Brief Review of Bioinformatics Tools for Glycosylation Analysis by Mass Spectrometry

“…e disadvantage of GlycoMiner is that the glycan compositions assignments when the quality the spectra input is low. 33) "Protein Prospector" combines CID and ETD search results into a single output le for glycopeptide identi cation by a database search. In addition, the so ware permits a manual comparison of the potential site assignments and the annotation of the glycosylated and de-glycosylated fragment ions in the same spectrum, which is a particularly useful feature for CID spectra of O-linked glycopeptides, where both fragment types are generally present.…”