“Comparative Analysis of Glycoproteomic Software Using a Tailored Glycan Database”

Glycoproteomics is a rapidly developing field, and data analysis has been stimulated by several technological innovations. As a result, there are many software tools from which to choose; and each comes with unique features that can be difficult to compare. This work presents a head-to-head comparison of five modern analytical software: Byonic, Protein Prospector, MSFraggerGlyco, pGlyco3, and GlycoDecipher. To enable a meaningful comparison, parameter variables were minimized. One potential confounding variable is the glycan database that informs glycoproteomic searches. We performed glycomic profiling of the samples and used the output to construct matched glycan databases for each software. Up to 19,000 glycopeptide spectra were identified across three replicates of wild-type SH-SY5Y cells. There was substantial overlap among most software for glycoproteins identified, locations of glycosites, and glycans, although Byonic reported a suspiciously large number of glycoproteins and glycosites of questionable reliability. We show that Protein Prospector identified the most glycopeptide spectrum matches with high agreement to known glycosites in UniProt. Overall, our results indicate that glycoproteomic searches should involve more than one software to generate confidence. It may be useful to consider software with peptide-first approaches and with glycan-first approaches.