Probing arginine-phosphopeptide interactions in non-covalent peptide-peptide ion complexes using gas-phase cross-linking and Born-Oppenheimer molecular dynamics calculations

Abstract

We report a study of non-covalent complexes of phosphopeptides pXAAAA and N-Ac-pXAAAA (X = Ser, Thr, Tyr) with arginine-containing peptides carrying diazirine 4,4-azipentyl tags at the N-terminus, *LGG(A)nR, or in the photoleucine residue, L*GG(A)nR (n = 3–5). Complexes with *LGG(A)nR were successfully generated as singly charged ions in the gas phase in 0.6–3.5% yields. In contrast, complexes with L*GG(A)nR were formed in negligible (<0.1%) yields. Selective photodissociation at 355 nm of the diazirine ring in [*LGG(A)nR + pXAAAA + H]+ ions resulted in loss of N2, producing large (75–95%) fractions of non-dissociating complexes that were further probed by collision-induced dissociation tandem mass spectrometry (CID-MS3). Covalent cross-links in the complexes produced by photoinduced carbene insertion were identified by specific CID-MS3 dissociations involving H3PO4 transfer and backbone fragmentations. N-acetylation in the phosphopeptides was found to have a substantial negative effect on the formation of covalent cross-links. Amongst the phosphorylated amino acid residues, pTyr showed the highest tendency (up to 92%) to form covalent cross-links. The fractions of covalent cross-links substantially increased with the length of the photopeptide chain. Born-Oppenheimer molecular dynamics (BOMD) calculations of canonical and zwitterionic protomers of the (pYAAAA + *LGGAAAR + H)+ complex indicated multiple close contacts between the incipient carbene of the diazirine ring and the XH bonds (XC, N, O) in the phosphopeptide. BOMD in combination with structural analysis by density functional theory calculations were used to interpret the experimental data and explain the cross-linking efficiencies.