Unifying N- and O-glycopeptide dissociation methods with expedient electron transfer dissociation
The glycoproteome is highly heterogeneous, where each glycosite can be modified by potentially hundreds of glycan compositions. This complexity necessitates methods that can provide glycosite-specific characterization in a high-throughput manner. Electron transfer dissociation (ETD) is particularly valuable for glycopeptide analysis, where it is critical for glycosite localization in O-glycopeptides and often produces higher quality spectra than higher energy collisional dissociation (HCD) for N-glycopeptides. Despite this, lower quality HCD spectra are often favored in high-throughput glycoproteomics experiments because the near instantaneous fragmentation of HCD results in faster MS/MS spectral acquisition relative to ETD. In short, overhead times for ETD reactions limit acquisition rates and sensitivity that can outweigh its advantages. Here we implement changes to ion handling, scan parallelization, and ion-ion reaction conditions to increase ETD MS/MS acquisition rates on the Orbitrap Tribrid MS platform. We explore fundamental parameters of ETD ion-ion reactions, including reaction q-values (i.e., Mathieu q-values) and ion-ion reaction time, showing that we can reduce the overhead of ion-ion reaction scan functions and improve the overall synchronization of data acquisition. These optimizations lead to faster spectral acquisition times for ETD MS/MS spectra while maintaining high spectral quality. Ultimately, these improvements allow the ETD scan functions to be nested within ion accumulation times needed per MS/MS spectrum, enabling ETD spectral acquisition to be parallelized within the duty cycle of the instrument and removing the major time cost to doing ETD-based fragmentation. We argue that this expedient implementation of ETD makes ETD-based fragmentation a universally beneficial dissociation method for both N- and O-glycopeptides.