Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers. (21st November 2018)

• Record Type: Journal Article
• Authors: Horne, Jim E.; Walko, Martin; Calabrese, Antonio N.; Levenstein, Mark A.; Brockwell, David J.; Kapur, Nikil; Wilson, Andrew J.; Radford, Sheena E.
• Is Part Of: Angewandte Chemie. Volume 130:Number 51(2018)
• Page Start: 16930
• Page End: 16934
• Publication Date: 2018-11-21
• Subjects: Chemische Vernetzung -- Diazoverbindungen -- Massenspektrometrie -- Photoaffinitätsmarkierung -- Protein-Protein-Wechselwirkungen; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201809149
• Languages: English
• ISSNs: 0044-8249

Abstract

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). Abstract : Schnelle Photovernetzung : Photovernetzende Reagenzien mit Methanthiosulfonat‐ und Diazirinfunktionalitäten wurden entwickelt. Nach Vernetzung und Reduktion bleibt ein Thiol‐Tag an der Interaktionsstelle, der mittels Massenspektrometrie lokalisiert und zur Anreicherung auch von Vernetzungen mit geringer Häufigkeit verwendet wird. Eine UV‐LED‐Bestrahlungsplattform reduziert die Vernetzungszeiten auf 10 s.