Issue 7, 2023

Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF

Abstract

Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.

Graphical abstract: Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF

Supplementary files

Article information

Article type
Paper
Submitted
13 Oct 2022
Accepted
28 Feb 2023
First published
06 Mar 2023
This article is Open Access
Creative Commons BY license

Analyst, 2023,148, 1534-1542

Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF

K. A. Graham, C. F. Lawlor and N. B. Borotto, Analyst, 2023, 148, 1534 DOI: 10.1039/D2AN01682F

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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