Photocaged Quinone Methide Crosslinkers for Light‐Controlled Chemical Crosslinking of Protein–Protein and Protein–DNA Complexes

Abstract

Small‐molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo‐crosslinkers target almost all residues non‐specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)‐based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein–protein and protein–DNA networks and for structural biology.

Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers

“…Various types of photoreactive reagents have been explored in XL-MS studies (13,(22)(23)(24)(25)(26)(27)(28)(29)(30), J o u r n a l P r e -p r o o f almost all of which have been heterobifunctional cross-linkers with an amine-reactive specific end and a nonspecific end. Among the commonly used photoreactive groups, alkyl diazirine is most attractive due to its small size, long excitation wavelength, photostability, reactivity, and proven success in XL-MS studies (22,(24)(25)(26)(27)(28)(29)(30). Diazirines are activated by UV light to yield highly reactive carbenes, which then react with an X-H bond (X: C, N, O, S) of any proximal amino acids (24,25,27,(29)(30)(31).…”

“…Diazirines are activated by UV light to yield highly reactive carbenes, which then react with an X-H bond (X: C, N, O, S) of any proximal amino acids (24,25,27,(29)(30)(31). While promising, the indiscriminate nature of photocrosslinking often results in highly complex and low abundance cross-linked products that complicate MS analysis and database searching, thus limiting its application predominantly to single proteins (24)(25)(26)(27)(28)30). Therefore, to advance photo-reactive XL-MS studies for complex PPI mapping, it is essential to develop novel reagents that permit effective MS detection and accurate identification of photocross-linked peptides.…”

Photocatalytic Proximity Labeling for Profiling the Subcellular Organization of Biomolecules

“…Upon the irradiation of blue light (450 nm), the Ir complex catalyzes the transformation of aryl azide to aniline, which is followed by the rapid decaging of para‐azidobenzyl group to unveil a reactive quinone methide intermediate (Figure 2a). As a Michael acceptor, quinone methide primarily targets amino acid residues with nucleophilic side‐chains, including lysine, serine, etc [25] . Owing to the selective accumulation of Ir complexes in the mitochondria, CAT‐Prox allows profiling mitochondrial proteome dynamics in macrophages upon inflammatory stimulation [24] …”

Recent Advances in Quinone Methide Chemistry for Protein-Proximity Capturing

“…In 2019, Wang and co-workers 17 reported a photocaged quinone methide (PQM)-based probe that target nine nucleophilic residues through Michael addition. The residues, including Gln, Arg, and Asn, are not easily subjected to other reported probes.…”