Increased Single-Spectrum Top-Down Protein Sequence Coverage in Trapping Mass Spectrometers with Chimeric Ion Loading

Abstract

Fourier transform mass spectrometers routinely provide high mass resolution, mass measurement accuracy, and mass spectral dynamic range. In this work, we utilize 21 T Fourier transform ion cyclotron resonance (FT-ICR) to analyze product ions derived from the application of multiple dissociation techniques and/or multiple precursor ions within a single transient acquisition. This ion loading technique, which we call, "chimeric ion loading", saves valuable acquisition time, decreases sample consumption, and improves top-down protein sequence coverage. In the analysis of MCF7 cell lysate, we show collision-induced dissociation (CID) and electron-transfer dissociation (ETD) on each precursor on a liquid chromatography-mass spectrometry (LC-MS) timescale and improve mean sequence coverage dramatically (CID-only 15% vs chimeric 33%),

Sequential Ion–Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

“…These numbers increase to 18, 16, 25, and 52 considering the 6 × 250 PTCR MS 3 experiments (see panels E of Figures –). This observation corroborates the notion of complementarity between radical-driven ion activation methods and vibrational energy threshold ion activation techniques such as collision-induced dissociation and higher-energy collisional dissociation (HCD), which had been explored and demonstrated by multiple studies, even using the same standard proteins chosen for the present work. , …”

“…This observation corroborates the notion of complementarity between radical-driven ion activation methods and vibrational energy threshold ion activation techniques such as collisioninduced dissociation and higher-energy collisional dissociation (HCD), which had been explored and demonstrated by multiple studies, even using the same standard proteins chosen for the present work. 32,58 Given the popularity of HCD in top-down proteomics, we also tested this fragmentation method and compared HCD MS 2 results with HCD MS 2 -1 × 1800 PTCR MS 3 data acquisition. For each of the 12 total protein precursor ions investigated (three different charge states for each protein), PTCR allowed the identification of additional fragments and increased the matched fragments' median mass, not only leading to higher sequence coverages than the corresponding MS 2 experiments but also providing better characterization of the central portions of the proteins' sequences (Figures S13− S16).…”

Sequential Ion-Ion Reactions for Enhanced Gas-Phase Sequencing of Large Intact Proteins in a Tribrid Orbitrap Mass Spectrometer

“…This observation corroborates the notion of complementarity between radical-driven ion activation methods and vibrational energy threshold ion activation techniques such as collisioninduced dissociation and higher-energy collisional dissociation (HCD), which had been explored and demonstrated by multiple studies, even using the same standard proteins chosen for the present work. 31,52 Given the popularity of HCD in top-down proteomics, we also tested this fragmentation method and compared HCD MS 2 results with HCD MS 2 -1x1800 PTCR MS 3 data acquisition. For each of the 12 total protein precursor ions investigated (three different charge states for each protein), PTCR allowed the identification of additional fragments and increased the matched fragments' median mass, not only leading to higher sequence coverages than the corresponding MS 2 experiments but also providing better characterization of the central portions of the proteins' sequences ( Figure S13-S16).…”

Characterization of Structural Hemoglobin Variants by Top-Down Mass Spectrometry and R Programming Tools for Rapid Identification

“…Although chimeric ion loading is not implemented on any commercially available mass spectrometry instrument, its use enabled consideration of a wider array of diagnostic ions for accurate variant identification and provided a rationale for development of data analysis tools that support electron- and collision-based dissociation techniques. It also reduced sample consumption, data acquisition, and analysis times by a factor of 2 relative to the performance of separate ETD and CID MS/MS experiments. , …”

“…24 Weisbrod et al recently introduced "chimeric ion loading", in which CID and ETD product ions derived from separate "batches" of precursor ions are accumulated in an external multipole storage device (MSD) and injected together into the ICR cell for high-resolution mass analysis. 29 In this way, both b-/y-and c-/z • -ions can be detected simultaneously in a single time-domain transient. Here, the "chimeric ion loading" technique is applied to diagnose Hb β variants.…”