Ion/ion reactions and instrument development for the modification of gas phase bio-ions

Several gas phase ion/ion reactions directed toward the modification of biologically relevant ions are discussed herein. These modifications range from gas phase conjugations of various R groups, both specific and general fashions, to the generation of radical cationic species. Carbodiimide functionalities were shown to exhibit selectivity towards carboxylic acids in the gas phase, forming what is suspected to be an amide bond as a novel gas phase reaction product. Variations of the reaction conditions were tested to specifically elucidate the reactive moieties. Multiple carbodiimide-containing reagents were used to demonstrate the versatility of the reaction, suggesting this chemistry can be used to append different functional groups to carboxylic acid sites. Ion/Ion reactions were also utilized to observe the alkylation of multiple anion types, including carboxylates, sulfonates, phosphates and alkoxides. Ammonium-, phosphonium- and sulfonium-centered reagents were studied experimentally to evaluate alkylation efficiency, while DFT calculations were employed to rationalize empirical results. This alkylation was demonstrated using varying lengths of alkyl chains as well as more interesting R groups, including amino groups, benzyl groups and quaternary functionalities, suggesting that the reactivity of ammonium and sulfonium reagents with a range of anions may be utilized as a versatile conjugation method in the gas phase. Novel methods were developed for the generation of radical peptide cations in the gas phase. This was achieved by combining collisional activation or ultraviolet photodissociation with ion/ion reactions. Anions containing azo functionalities underwent hemolytic cleavage upon collisional activation to yield initial odd electron species that would interact with multiply protonated peptides and proteins either through electrostatic interactions (i.e. the formation of an electrostatic complex) or through gas-phase covalent additions through N-hydroxysuccinimide reactions. Both of these methods for introducing radical sites to the cationic species facilitated hydrogen abstraction from the analyte, and radical-directed dissociation of peptide bonds and side chains was ultimately observed. Similar methods were employed using the selective ultraviolet photodissociation of carbon-iodine bonds rather than collisional activation of azo groups. In these instances, ion/ion reactions were used to introduce anions containing carbon-iodine bonds to multiply protonated peptides, either through solely electrostatic interactions, or through covalent addition using N-hyroxysuccinimide reactivity. These carbon-iodine bonds were then homolytically cleaved using 266 nm photons to generate the initial radical site, which proceeded to abstract a hydrogen from the cationic peptide. This also ultimately resulted in the observation of radical-directed dissociation