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

AI Article Synopsis

Article Abstract

Obtaining extensive sequencing of an intact protein is essential in order to simultaneously determine both the nature and exact localization of chemical and genetic modifications which distinguish different proteoforms arising from the same gene. To effectively achieve such characterization, it is necessary to take advantage of the analytical potential offered by the top-down mass spectrometry approach to protein sequence analysis. However, as a protein increases in size, its gas-phase dissociation produces overlapping, low signal-to-noise fragments. The application of advanced ion dissociation techniques such as electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) can improve the sequencing results compared to slow-heating techniques such as collisional dissociation; nonetheless, even ETD- and UVPD-based approaches have thus far fallen short in their capacity to reliably enable extensive sequencing of proteoforms ≥30 kDa. To overcome this issue, we have applied proton transfer charge reduction (PTCR) to limit signal overlap in tandem mass spectra (MS) produced by ETD (alone or with supplemental ion activation, EThcD). Compared to conventional MS experiments, following ETD/EThcD MS with PTCR MS prior to / analysis of deprotonated product ions in the Orbitrap mass analyzer proved beneficial for the identification of additional large protein fragments (≥10 kDa), thus improving the overall sequencing and in particular the coverage of the central portion of all four analyzed proteins spanning from 29 to 56 kDa. Specifically, PTCR-based data acquisition led to 39% sequence coverage for the 56 kDa glutamate dehydrogenase, which was further increased to 44% by combining fragments obtained via HCD followed by PTCR MS.

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http://dx.doi.org/10.1021/jasms.1c00062DOI Listing

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