Solution versus gas-phase modification of peptide cations with NHS-ester reagents.

A comparison between solution and gas phase modification of primary amine sites in model peptide cations with N-hydroxysuccinimide (NHS) ester reagents is presented. In all peptides, the site of modification in solution was directed to the N-terminus by conducting reactions at pH=5, whereas for the same peptides, a lysine residue was preferentially modified in the gas phase. The difference in pKa values of the N-terminus and ε-amino group of the lysine allows for a degree of control over sites of protonation of the peptides in aqueous solution. With removal of the dielectric and multiple charging of the peptide ions in the gas phase, the accommodation of excess charge can affect the preferred sites of reaction. Interaction of the lone pair of the primary nitrogen with a proton reduces its nucleophilicity and, as a result, its reactivity towards NHS-esters. While no evidence for reaction of the N-terminus with sulfo-NHS-acetate was noted in the model peptide cations, a charge inversion experiment using bis[sulfosuccinimidyl] suberate, a cross-linking reagent with two sulfo-NHS-ester functionalities, showed modification of the N-terminus. Hence, an unprotonated N-terminus can serve as a nucleophile to displace NHS, which suggests that its lack of reactivity with the peptide cations is likely due to the participation of the N-terminus in solvating excess charge.