Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents

Abstract

A major challenge in cross-linking/mass spectrometry (MS) is targeting carboxyl functions in proteins under physiological conditions that do not disturb the protein's conformation. Cross-linking of glutamic acid and aspartic acid residues in proteins will greatly expand the scope of structural mass spectrometry. We discovered that carboxyl-reactive cross-linkers have already been employed for many years in cross-linking/MS studies, yet in a completely different context. Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine cross-linkers, are currently considered to react nonspecifically upon UV-A photoactivation with all 20 proteinogenic amino acids through a reactive carbene that inserts mainly into C-H bonds. We discovered that the cross-linking capability of diazirines based on X-H (X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines isomerize to linear diazo compounds that can react with carboxylic acids to yield esters. On top of that, the resulting cross-linked products are MS-cleavable allowing an automated analysis of cross-links via customized software tools. Therefore, diazirines open an entirely new route for photo-cross-linking of carboxylic acids. Previous cross-linking studies using diazirines have to be revisited in the light of these findings.

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7

“…Analysis of irradiated and non-irradiated mixtures of the photoprobe with VSD2-NavAb was performed using MeroX 1.6.6, a software originally designed to identifying crosslinking sites between peptides that result from a MS 2 -cleavable crosslinker (Iacobucci et al, 2018), and based on StavroX 3.6.6, which has been used previously in crosslinking studies (Gö tze et al, 2012). In the case of diazirine photoactivation, the diazo reactive species often reacts with the carboxylic acid in glutamic and Schematic of VSD2-NavAb channel structure and partial amino acid sequence.…”

Free radical‐initiated peptide sequencing (FRIPS)‐based cross‐linkers for improved peptide and protein structure analysis

“…Chemical cross‐linking combined with MS has matured into an alternative method to NMR and X‐ray crystallography for deriving three‐dimensional structures of proteins and protein assemblies . The introduction of covalent bonds between specific amino acid side chains via a linker molecule presents the principle of chemical cross‐linking and is the fundament for structure‐based MS.…”

“…The obtained distance constraints serve as basis for generating three‐dimensional structural models of proteins and protein complexes by computational modeling . During the last years, chemical cross‐linking/MS has demonstrated its power for the 3D‐structure analysis of numerous proteins and protein complexes, comprising even very large (MDa) assemblies and the number of publications for solving important questions in structural biology has steadily increased.…”

“…4 | THE CROSS-LINKING/MS APPROACH Chemical cross-linking combined with MS has matured into an alternative method to NMR and X-ray crystallography for deriving three-dimensional structures of proteins and protein assemblies. [102][103][104][105][106][107][108] The introduction of covalent bonds between specific amino acid side chains via a linker molecule presents the principle of chemical cross-linking and is the fundament for structure-based MS. The by far most popular reagents are homobifunctional, amine-reactive cross-linkers, mainly N-hydroxysuccinimide (NHS) esters.…”

“…Even-electron fragmentation pathway of the TEMPO-Bz-linker in (+)ESI-CID-MS/MS in case the protonation occurs at the TEMPO-Bz moiety itself (with peptide1 = R1 and peptide2 = R2). 97 linking/MS has demonstrated its power for the 3D-structure analysis of numerous proteins 107 and protein complexes, comprising even very large (MDa) assemblies [108][109][110] and the number of publications for solving important questions in structural biology has steadily increased.…”