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

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.