Carbene Cross-Linking in Gas-Phase Peptide Ion Scaffolds

Abstract

Scaffolds consisting of a peptide, a phthalate linker, and a 4,4-azipentyl group were synthesized and used to study intramolecular peptide–carbene cross-linking in gas-phase cations. Carbene intermediates were generated by UV-laser photodissociation at 355 nm of the diazirine ring in mass-selected ions, and the cross-linked products were detected and quantified by collision-induced dissociation tandem mass spectrometry (CID-MS n , n = 3–5). Peptide scaffolds containing Ala and Leu residues with a C-terminal Gly gave 21–26% yields of cross-linked products, while the presence of the Pro and His residues decreased the yields. Experiments using hydrogen–deuterium–hydrogen exchange, carboxyl group blocking, and analysis of CID-MS n spectra of reference synthetic products revealed that a significant fraction of cross-links involved the Gly amide and carboxyl groups. Interpretation of the cross-linking results was aided by Born–Oppenheimer molecular dynamics (BOMD) and density functional theory calculations that allowed us to establish the protonation sites and conformations of the precursor ions. Analysis of long (100 ps) BOMD trajectories was used to count close contacts between the incipient carbene and peptide atoms, and the counting statistics was correlated with the results of gas-phase cross-linking.

Covalent crosslinking in gas-phase biomolecular ions. An account and perspective

“…This has allowed us to correlate the computationally determined complex structures and their potential for amino acid cross linking with experimental results obtained by photodissociation and product sequence analysis by CID-MS 3 . 38,[65][66][67][68][69][70][71][72] Whereas carbene reactions with hydrocarbons have been studied in quite a detail, the effect of polar groups on the interaction with carbenes has received less attention and has been focused on small solvent molecules, such as water and alcohols. Experimental studies of peptide crosslinking in solution have chiefly been of a heuristic nature.…”

“…In these synthetic constructs, the peptide and diazirine carrier, such as the 4,4-azi pentyl group, are mounted on a covalent scaffold that limits the conformational motion of both moieties. 72 Photochemical crosslinking between the carbene intermediate and peptide bonds is detected by CID-MS 3 analysis of the products. A scaffold of the Ala-Ala-Leu-Gly tetrapeptide with the phthalate linker is shown in Scheme 5.…”

“…38,66 Scaffolds with AAPG and AAHG peptides gave lower yields. 72 The C-terminal carboxyl was found to play an important role in crosslinking, as established by carboxyl blocking as an ester and by H/D exchange. The H/D exchange experiment consisted of exhaustive exchange of all active protons in the AALG scaffold into the D cycle, followed by photodissociation.…”

“…The structure assignment of the photoproduct was finalized by CID-MS 2 of an authentic macrocyclic lactone that was synthesized, and its spectrum showed the same features as that of the photoproduct. 72 This way, the resolution in the product structure analysis was augmented to achieve identification of individual amino acid bonds that were involved in crosslinking.…”

“…This has allowed us to correlate the computationally determined complex structures and their potential for amino acid cross linking with experimental results obtained by photodissociation and product sequence analysis by CID-MS 3 . 38,65–72…”