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Characterization of Protein Structure with Ion Mobility Mass Spectrometry, Multiplexed Fragmentation Strategies and Data Directed Analysis

preprint
submitted on 08.11.2020, 16:41 and posted on 10.11.2020, 05:39 by Rachelle Black, Alexey Barkhanskiy, Lennart Ramakers, Alina Theisen, Jeffery Brown, Bruno Bellina, Drupad Trivedi, Perdita Barran

Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without a priori assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.

Funding

BB/L002655/1, BB/L016486/1 and BB/M01108/1; MS SPIDOC project funded by the European Union’s Horizon 2020 FET-OPEN research and innovation programme (Grant agreement No. 801406); EP/S005226/1

History

Email Address of Submitting Author

perdita.barran@manchester.ac.uk

Institution

The University of Manchester

Country

United Kingdom

ORCID For Submitting Author

0000-0002-7720-586X

Declaration of Conflict of Interest

No coi

Version Notes

version 1.0

Exports