Intra- and Inter-Molecular Cross-Linking of Peptide Ions in the Gas Phase: Reagents and Conditions

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.