Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer

Varun V. Gadkari, Brock R. Juliano, Christopher S. Mallis, Jody C. May, Ruwan T. Kurulugama, John C. Fjeldsted, John A. McLean, David H. Russell, Brandon T. Ruotolo

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9 Scopus citations


Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established. Here we evaluate the reproducibility of the CIU data produced across three laboratories (University of Michigan, Texas A&M University, and Vanderbilt University). CIU data were collected for a variety of protein ions ranging from 8.6-66 kDa. Within the same laboratory, the CIU fingerprints were found to be repeatable with root mean square deviation (RMSD) values of less than 5%. Collision cross section (CCS) values of the CIU intermediates were consistent across the laboratories, with most features exhibiting an interlaboratory reproducibility of better than 1%. In contrast, the activation potentials required to induce protein CIU transitions varied between the three laboratories. To address these differences, three source assemblies were constructed with an updated ion activation hardware design utilizing higher mechanical tolerance specifications. The production-grade assemblies were found to produce highly consistent CIU data for intact antibodies, exhibiting high precision ion CCS and CIU transition values, thus opening the door to establishing databases of CIU fingerprints to support future biomolecular classification efforts.

Original languageEnglish (US)
Pages (from-to)391-401
Number of pages11
Issue number2
StatePublished - Dec 13 2022
Externally publishedYes

Bibliographical note

Funding Information:
V. V. G., B. R. J., B. T. R. acknowledge support from Agilent Technologies Thought Leader Award and University Relations grant programs. CIU method development in the Ruotolo laboratory is supported through an Agilent Technologies Applications and Core Technology University Research (ACT-UR) Grant. C. S. M. and D. H. R. acknowledge support from NIH grants R01GM121751 and P41GM1285771. J. C. M. and J. A. M. acknowledge the National Institutes of Health, National Institute of Child Health and Human Development (NIH HHS, R01HD102752) for funding support as well as the Center for Innovative Technology (CIT) at Vanderbilt for providing resource support for the experiments conducted at Vanderbilt University.

Publisher Copyright:
© 2023 The Royal Society of Chemistry

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