Improvements in Glycoproteomics through Architecture Changes to the Orbitrap Tribrid MS Platform

Hardware changes introduced on the Orbitrap Ascend Tribrid MS include dual ion routing multipoles (IRMs) that enable parallelized accumulation, dissociation, and Orbitrap mass analysis of three separate ion populations. The balance between these instrument functions is especially important in glycoproteomics, where complexities of glycopeptide fragmentation necessitate large precursor ion populations and long ion accumulation times for quality MS/MS spectra. To compound matters further, dissociation methods like electron transfer dissociation (ETD) that benefit glycopeptide characterization come with overhead times that slow down scan acquisition. Here we explored how the Orbitrap Ascend's dual IRM architecture can improve glycopeptide analysis, with a focus on O-glycopeptide characterization using ETD with supplemental collisional activation (EThcD). We found that parallelization of ion accumulation and EThcD fragmentation increased scan acquisition speed without sacrificing spectral quality, subsequently increasing the number of O-glycopeptides identified relative to analyses on the Orbitrap Eclipse (i.e., the previous generation Tribrid MS). Additionally, we systematically evaluated ion-ion reaction times and supplemental activation energies used for EThcD to understand how best to utilize acquisition time. We observed that shorter-than-expected ion-ion reaction times minimized scan overhead time without sacrificing <i>c</i>/<i>z</i>•-fragment ion generation and that higher supplemental collision energies can generate combinations of glycan-retaining and glycan-neutral-loss peptide backbone fragments that benefit O-glycopeptide identification. We also saw improvements in N-glycopeptide analysis using collision-based dissociation, especially with methods using faster scan speeds. Overall, these data show how architectural changes to the Tribrid MS platform benefit glycoproteomic experiments by parallelizing scan functions to minimize overhead time and improve sensitivity.