Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

Abstract

Abstract. Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N 2 from the L* diazirine rings underwent insertion into X−H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13 C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H) + ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H] + ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide-peptide ion interactions in the gas phase.

Photoleucine Survives Backbone Cleavage by Electron Transfer Dissociation. A Near-UV Photodissociation and Infrared Multiphoton Dissociation Action Spectroscopy Study

“…The gas-phase chemistry of photoleucine and photomethionine-containing peptide ions has been studied with the aim to understand their properties [3,4] and exploit their photochemistry for peptide-ligand footprinting in gas-phase ion-molecule complexes [5,6]. Collisioninduced dissociation under slow heating conditions of singly and doubly charged L*-and M*-tagged peptide ions proceeds by competing loss of N 2 and backbone dissociations [3].…”

Position‐tunable diazirine tags for peptide–peptide ion cross‐linking in the gas phase

“…Cross‐linking using L*‐tagged and M*‐tagged peptides in solution is known to achieve only low yields of identifiable products . In contrast, UV photodissociation of mass‐selected ion‐molecule complexes (UVPD‐MS 2 ) and the ensuing intramolecular bond insertion reactions have been reported to proceed with up to 30–40% yields of covalent products that can be further analyzed by tandem mass spectrometry after ion mass separation and collisional excitation in an ion trap (CID‐MS 3 ) …”

“…Previous research of gas‐phase cross‐linking in peptide–peptide ion complexes used synthetic peptides that were photolabeled with L* in specific sequence positions . Sequence analysis of the cross‐linked peptides, combined with Born–Oppenheimer molecular dynamics, indicated that the non‐covalent interactions in the complexes were chiefly due to hydrogen bonding of charged and polar groups whereas the nonpolar L* side chains were not involved.…”

“…Gas‐phase cross‐linking is illustrated with non‐covalent complexes of GLGGK* with a library of six nonpolar pentapeptides. The amino acid sequence of the photo‐tagged peptide was chosen to allow us to make a comparison with the results previously obtained for GL*GGK, a photoleucine containing peptide, and nonbasic target peptides such as GLLLG (Table ) . The [GAMVG + GLGGK* + H] + ion complex was formed by electrospray ionization (Fig.…”

“…The backbone fragment ions in Fig. (c) are denoted according to the nomenclature introduced previously, whereby capital letters (M*, Y, B) refer to the photo‐tagged peptide and its residues, and small letters (m, b, y) refer to the target peptide. The B, Y, b, and y fragment ion nomenclature follows that of Roepstorff–Fohlman–Biemann for regular peptides .…”