GlycoHybridSeq: Automated Identification of N-Linked Glycopeptides Using Electron Transfer/High-Energy Collision Dissociation (EThcD)

[Image: see text] Glycosylation is one of the most common post-translational modifications (PTM) occurring in a large variety of proteins with important biological functions in human and other higher organisms. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been routinely used to characterize site-specific protein glycosylation at high throughput in complex glycoproteomic samples. Recently, electron transfer/high-energy collision dissociation (EThcD) was introduced for glycopeptide identification, which offers rich structural information on glycopepides with the fragment ions from the cleavages of both the glycan and the peptide backbone. Herein, we present the software GlycoHybridSeq for automated interpretation of EThcD-MS/MS spectra from glycoproteomic data using a customized scoring function, which enables the functionalities of identifying glycopeptides, characterizing glycosylation sites, and distinguishing some isomeric glycans. We evaluate GlycoHybridSeq on glycoproteomic data collected for cancer biomarker discovery. The results showed that it achieved comparable or better performance than that of Byonic and MSFragger. GlycoHybridSeq is released as an open source software and is ready to be used in large-scale glycoproteomic data analyses.