Electron-Activated Dissociation and Collision-Induced Dissociation Glycopeptide Fragmentation for Improved Glycoproteomics

Abstract Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterisation of site-specific glycosylation of proteins remains analytically challenging. Collision induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but loss of labile modifications render CID inappropriate for detailed characterisation of site-specific glycosylation. Electron-based dissociation (ExD) methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localisation, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O - and N -glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localisation. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localisation confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterisation.