Front-End Electron Transfer Dissociation: A New Ionization Source

Abstract

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion–ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

“…Unless otherwise stated, all MS analyses were carried out on an in-house modified (21) hybrid Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, CA). In the time that the experiments described below were performed, the instrument was upgraded to include a high-field Orbitrap mass analyzer (26).…”

“…Velos Pro TM was modified to introduce reagent anions from the front by adding a glow discharge ionization source between the stacked ring ion guide and multipole 00, as described previously (21). The discharge is used to ionize ETD and IIPT reagents (fluoranthene and sulfur hexafluoride, respectively) for ion/ion reactions.…”

“…We have previously described modifications to a Thermo Fisher Scientific Orbitrap Velos Pro TM mass spectrometer that make it possible to perform multiple fills of the C-trap with product ions from sequential ion/ion reactions of intact proteins prior to high resolution mass analysis (1,21). The result is a dramatic enhancement of the observed fragment ion current and significant improvement of signal-to-noise ratios (SNRs) in MS/MS spectra.…”

Evolution of Orbitrap Mass Spectrometry Instrumentation

“…The Orbitrap Fusion instrument incorporates a novel ETD reagent ion source, utilizing Townsend discharge for ionization of the reagent molecules, which significantly reduces maintenance and tuning requirements (84) relative to the earlier filament-based ETD ionization source. The inclusion of a novel option for preferential selection of higher charge ions with lower m/z allows the Orbitrap Fusion instrument to target ions, in a data-dependent fashion, known to be more susceptible to ETD fragmentation, thereby potentially increasing the identification rate (83).…”

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis

“…The ETD hardware is adapted from the Thermo Fusion [23] and follows from the work of Earley et al [24] Fluoranthene reagent ions are generated by Townsend discharge in an excess of nitrogen gas, in an ionization volume located between the slens and the first quadrupole ion guide in the Velos Pro (termed Bfront-ETD^or BFETD^by the inventors). The analyte and reagent targets are 200,000 charges with a reaction period of 10 ms for dissociation of the 34+ charge state of carbonic anhydrase in the Velos Pro linear trap.…”