Horne, J, Walko, M, Calabrese, A et al. (5 more authors) (2018) Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers. Angewandte Chemie - International Edition, 57 (51). pp. 16688-16692. ISSN 1433-7851
Abstract
Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulphonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue‐unbiased diazirine‐based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait initially transfers a thiol‐containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well‐defined peptide/protein regulatory interaction (BID80‐102/MCL‐1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | chemical crosslinking; mass spectrometry; Diazo Compounds; Photoaffinity labeling; protein-protein interactions |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology 2 (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Mechanical Engineering (Leeds) > Institute of Engineering Thermofluids, Surfaces & Interfaces (iETSI) (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/K000659/1 BBSRC BB/M012573/1 BBSRC BB/N007603/1 EPSRC EP/N035267/1 Wellcome Trust 208385/Z/17/Z |
Depositing User: | Symplectic Publications |
Date Deposited: | 05 Nov 2018 11:38 |
Last Modified: | 25 Jun 2023 21:34 |
Status: | Published |
Publisher: | Wiley |
Identification Number: | 10.1002/anie.201809149 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:138143 |
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