In an article published in the Journal of the American Society for Mass Spectrometry, researchers demonstrated a novel segmented linear ion trap for multiple-stage tandem mass spectrometry. The external injection of reagent ions, photons, radical neutral species, collisions with neutrals, and electrons were all components of the ion activation network. The authors also highlighted the Omnitrap ion trap platform's design features in this paper.
The experimental research into the mobility of ions captured by rectangular waveforms was conducted by mapping the stability diagram, experimenting with various isolation methods, and tracing secular frequencies.
Three-stage (MS3) tandem mass spectrometry experiments were carried out efficiently. They integrated collision-induced dissociation of radical ions created by electron meta-ionization with two additional intermediate steps of accumulation and ion isolation. The multiple-stage tandem mass spectrometry produced an information-rich spectrum with signal-to-noise levels similar to those derived from a two-stage (MS2) mass spectrometry experiment.
While successfully integrating with time-of-flight analyzers, the ion trap platform was further improved by coupling to an Orbitrap mass analyzer.
Innovations in Mass Spectrometry
Mass spectrometry has become a crucial tool in bioanalytical chemistry research due to the introduction of soft ionization, improvements in fragmentation techniques, and the creation of high-performance mass analyzers.
Fragmentation instruments and methodologies must advance to improve the analysis of increasingly complex samples using mass spectrometry, where molecular identification and precise structure or sequence information are required.
In the Omnitrap platform, variations of every fragmentation technique mentioned so far were successfully used. The technology's unique multiple-stage tandem mass spectrometry capabilities and the new linear ion trap design features were outlined. This new device's main objective was to make high-performance top-down multiple-stage tandem mass spectrometry possible by giving users access to a broader ion activation network.
In addition to activation techniques based on external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals, the fragmentation toolbox was available for building multiple-stage tandem mass spectrometry studies included these techniques.
The Omnitrap platform consisted of three primary tandem processing of ions trapping areas formed by grouping nine successive quadrupole segments, from the entrance to the exit end, in a linear format. The quadrupole electrodes were mounted on ceramic platforms with pockets cut into them as part of a frame-ring arrangement. The total construction was put together with a 20 mm accuracy, and the electrodes' hyperbolic surface was wire-eroded to 5 mm.
Elevated pressure levels were preferred for effective thermalization of tandem processing of ions during their transfer between trapping zones. Such pressure levels were also necessary for axial injection of ions into the ion trap from an external source during collisional activation. An 85 L/s turbomolecular pump provided differential pumping, and insulators placed between the ceramic platforms hermetically sealed the ion trap chamber.
The Omnitrap platform was built with the efficient integration of current fragmentation techniques. It offered a flexible architecture for creating new tools for the tandem processing of ions and multiple-stage tandem mass spectrometry protocols. In top-down research, where in-depth characterization of intact proteins was desired, approaches, where different activation-dissociation stages were used consecutively to maximize the information output, were demonstrated to be necessary.
The MS-Product (Protein Prospector) online fragment calculator was changed to add multiple-charged internal band a-type ions for the data processing procedure, which was carried out using a new data processing tool created internally by Fasmatech.
New algorithms were created to fit the predicted isotopic distributions to ubiquitin fragmentation mass spectra based on several score functions to maximize the confidence in the assignments. The significant number of mistakes connected with identifying monoisotopic masses in highly congested spectra led to eliminating the deconvolution stage in this new data processing workflow.
Significance of the Study
The study's primary goal was to improve the ability of top-down multiple-stage tandem mass spectrometry to characterize intact biomolecules, especially in more effective systems where a diversified ion activation network could yield supplementary data.
By successfully achieving comprehensive sequence coverage for the ubiquitin 8+ charge state using four different fragmentation techniques employing electrons, photons, and reagent ions, the Omnitrap platform's ability to provide top-down multiple-stage tandem mass spectrometry was proved.
The Omnitrap was completed, and the unsaturated hydrocarbon outgassing from the polyetheretherketone (PEEK) material causing the increased background ion signal was also eliminated. A more straightforward graphical user interface was created to address the instrument control software's complexity.
The successful integration of the Omnitrap platform with the subsequent-generation Exploris Orbitrap mass spectrometers increased the device's versatility. Therefore, the Omnitrap platform qualified as a superb tool for investigating the gas-phase chemistry of ions throughout a broad mass range due to the combination of such a diversity of fragmentation techniques.
Papanastasiou, D., et al. (2022). The Omnitrap Platform: A Versatile Segmented Linear Ion Trap for Multidimensional Multiple-Stage Tandem Mass Spectrometry. Journal of the American Society for Mass Spectrometry. https://pubs.acs.org/doi/10.1021/jasms.2c00214?ref=pdf