Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

Abstract

Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately three fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

“…al. [36] , achieving maximum S/N is especially critical for top-down product ion mass spectra because the original precursor ion signal is distributed among many available fragmentation channels and fragment ion charge distributions. The two primary means for increasing S/N are spectral averaging and/or increasing the trapped ion population.…”

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry

“…Here we demonstrate that ETD outperformed CID with regard to confidence in protein identification and proteoform characterization (defined here with the scoring metrics, q -value, and C-score, respectively), which is consistent with prior studies. 14,24,26,30,31 However, a smaller precursor ion population is used, 9,23 and total ion current is diluted among more fragment ion channels, which requires more fragment ion fills of the MSD prior to high-resolution mass analysis. Ultimately, this results in a longer acquisition period required for ETD MS2 spectra.…”

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

“…39,41–46 We recently implemented AI-ETD on a quadrupole-Orbitrap-linear ion trap (q-OT-QLT, Orbitrap Fusion Lumos) and showed that it significantly improves peptide identifications and quality of fragmentation in ETD experiments (described in a companion manuscript). 47 With other ETD-specific improvements also incorporated on the Lumos platform, including a front-end ETD reagent source, 48 high capacity ETD for improved product ion signal-to-noise (S/N), 49 and calibrated ETD reaction times for optimal data acquisition in shotgun proteomic analyses, 50 this system represents the state-of-the art for ETD technology.…”