Olefinic reagents tested for peptide derivatization with switchable properties: Stable upon collision induced dissociation and cleavable by in‐source Paternò‐Büchi reactions

This contribution is part of our ongoing efforts to develop innovative cross‐linking (XL) reagents and protocols for facilitated peptide mixture analysis and efficient assignment of cross‐linked peptide products. In this report, we combine in‐source Paternò‐Büchi (PB) photo‐chemistry with a tandem mass spectrometry approach to selectively address the fragmentation of a tailor‐made cross‐linking reagent. The PB photochemistry, so far exclusively used for the identification of unsaturation sites in lipids and in lipidomics, is now introduced to the field of chemical cross‐linking. Based on trans‐3‐hexenedioic acid, an olefinic homo bifunctional amine reactive XL reagent was designed and synthesized for this proof‐of‐principle study. Condensation products of the olefinic reagent with a set of exemplary peptides are used to test the feasibility of the concept. Benzophenone is photochemically reacted in the nano‐electrospray ion source and forms oxetane PB reaction products. Subsequent CID‐MS triggered retro‐PB reaction of the respective isobaric oxetane molecular ions and delivers reliably and predictably two sets of characteristic fragment ions of the cross‐linker. Based on these signature ion sets, a straightforward identification of covalently interconnected peptides in complex digests is proposed. Furthermore, CID‐MSⁿ experiments of the retro‐PB reaction products deliver peptide backbone characteristic fragment ions. Additionally, the olefinic XL reagents exhibit a pronounced robustness upon CID‐activation, without previous UV‐excitation. These experiments document that a complete backbone fragmentation is possible, while the linker‐moiety remains intact. This feature renders the new olefinic linkers switchable between a stable, noncleavable cross‐linking mode and an in‐source PB cleavable mode.