Zwitterionic States in Gas-Phase Polypeptide Ions Revealed by 157-nm Ultra-Violet Photodissociation

A new method of detecting the presence of deprotonation and determining its position in gas-phase polypeptide cations is described. The method involves 157-nm ultra-violet photodissociation (UVPD) and is based on monitoring the losses of CO2 (44 Da) from electronically excited deprotonated carboxylic groups relative to competing COOH losses (45 Da) from neutral carboxylic groups. Loss of CO2 is a strong indication of the presence of a zwitterionic [(+)center dot center dot center dot(-)center dot center dot center dot(+)] salt bridge in the gas-phase polypeptide cation. This method provides a tool for studying, for example, the nature of binding within polypeptide clusters. Collision-activated dissociation (CAD) of decarboxylated cations localizes the position of deprotonation. Fragment abundances can be used for the semi-quantitative assessment of the branching ratio of deprotonation among different acidic sites, however, the mechanism of the fragment formation should be taken into account. Cations of Trp-cage proteins exist preferentially as zwitterions, with the deprotonation position divided between the Asp(9) residue and the C terminus in the ratio 3:2. The majority of dications of the same molecule are not zwitterions. Furthermore, 157-nm UVPD produces abundant radical cations M center dot+ from protonated molecules through the loss of a hydrogen atom. This method of producing M center dot+ ions is general and can be applied to any gas-phase peptide cation. The abundance of the molecular radical cations M center dot+ produced is sufficient for further tandem mass spectrometry (MS/MS), which, in the cases studied, yielded side-chain loss of a basic amino acid as the most abundant fragmentation channel together with some backbone cleavages.