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.