Horne, JE, Walko, M, Calabrese, AN et al. (5 more authors) (2018) Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers. Angewandte Chemie, 130 (51). pp. 16930-16934. ISSN 0044-8249
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 methanethiosulfonate (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 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: | Chemische Vernetzung; Diazoverbindungen; Massenspektrometrie; Photoaffinitätsmarkierung; Protein-Protein-Wechselwirkungen |
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) > Structural Molecular Biology 2 (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Mechanical Engineering (Leeds) The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 28 Oct 2019 10:54 |
Last Modified: | 28 Oct 2019 10:54 |
Status: | Published |
Publisher: | Wiley |
Identification Number: | 10.1002/ange.201809149 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:152661 |