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

“…In recent years, photochemistry has shown great potential in capturing regions inaccessible to residue-specific cross-linkers because of its nonspecific reactivity ( 2 , 3 , 20 , 21 ). Various types of photoreactive reagents have been explored in XL-MS studies ( 13 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ), 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 because of its small size, long excitation wavelength, photostability, reactivity, and proven success in XL-MS studies ( 22 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ).…”

“…Various types of photoreactive reagents have been explored in XL-MS studies ( 13 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ), 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 because of 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 photocross-linking 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 photoreactive 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.…”

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

“…27 This strategy has been used in inhibitors and prodrugs, [28][29][30] self-immolative linkers in dendrimers, 31 chemical selection of catalytic antibodies, 32,33 activity-based probes for various enzymes, including phosphatase, 34 glycosidases, 22,23,[35][36][37][38][39][40][41] , tyrosine phosphatase, 42 steroid sulfatase, 43 and beta-lactamase, 44 cell imaging 45,46 and probing protein-protein/DNA interactions. 47 Among these applications, the fluorine on either the monofluoromethyl or difluoromethyl group was used as a leaving group to form the active o-quinone methide intermediate via spontaneous elimination of a molecule of HF. However, the application of the quinone methide chemistry has been dwarfed by the dilemma that the monofluoromethyl group is more reactive and the protein-conjugated products are more stable than its difluoromethyl counterpart, but molecules containing a monofluoromethyl group suffer from poor stability in aqueous media, as the reactive monofluoromethyl group tends to be hydrolyzed by the surrounding water before the probe molecule reaches the active site of the target protein.…”

Trapping Transient RNA Complexes by Chemically Reversible Acylation

“…The majority of methods for studying nucleic acid interactions rely on chemical crosslinking. [22][23][24][25] The most widespread example of this is formaldehyde crosslinking, [26] which involves adducts to exocyclic amine groups; while it is routinely used for preserving RNA in clinical settings and for studying biomolecular interactions, [27] it suffers from low recovery yields. [27,28] Other amine-reactive aldehydic crosslinkers include 1,4-phenyl-diglyoxal.…”