Enhancing glycopeptide annotation and glycan localization using electron activated dissociation through a multiplexed parallel reaction monitoring design of experiments

Abstract

We present an optimized electron activated dissociation (EAD) methodology, based on hot electron capture dissociation, for liquid chromatography-tandem mass spectrometry characterization of N- and O-glycopeptides, using recombinant human erythropoietin as a model glycoprotein. Applying a full factorial design of experiments (DoE) approach, we first optimized LC-MS parameters (i.e., ion spray voltage, ion source temperature, and active gradient time) to enhance glycopeptide ionization efficiency while reducing in-source fragmentation. A second DoE was then applied to fine-tune EAD-specific parameters. Multiplexed parallel reaction monitoring was performed to efficiently and comprehensively optimize the electron beam current, reaction time, and electron kinetic energy of the EAD set-up. Finally, the optimized EAD parameters, initially determined using one glycoform per glycopeptide, were successfully applied in data-dependent acquisition mode to detect the overall glycoform composition of each studied glycopeptide. Byonic, Fragpipe and Mascot softwares, and several peak picking softwares were used to evaluate the potential of our optimized EAD set-up, and compare with collision induced dissociation (CID). The results confirmed that EAD improved confidence in glycan localization, while CID enabled the identification of a greater number of glycoforms but with less confident glycan assignments.