Ion mobility spectrometry (IMS) is a fast growing technique which can be coupled with computational methods to elucidate properties of gas-phase ions. Proteomics, the study of proteins and their functions, is fundamentally tied to the analytical chemistry of mass spectrometry. Proteins are digested into smaller peptides before being introduced into a mass spectrometer, where the peptides are fragmented into a series of product ions. In the present thesis, IMS was used in conjunction with computational theory to investigate the structures of product ions, the smallest of these product ions being known as b2 ions. The IMS technique used here is travelling wave IMS (TWIMS), a technique that requires regular calibration to determine individual ion mobilities and a common calibrant is polyalanine. This presents a problem as this study shows that the number of chiral centres present in polyalanine can lead to multiple different ion mobilities being detected if the standard mixture is not isomerically pure. This study aims to answer the questions of how well these techniques combine, how accurate are the parameters set in Mobcal (the programme used to compute theoretical CCS values), and if polyalanine is a suitable calibrant.
Computed models of b2 ions are presented with collisional cross sections (CCS) calculated via the software Mobcal. Mobcal is found not to contain suitable parameters for nitrogen drift gas calculations and will require further development. A variety of polyalanine isomers are synthesised and their ion mobilities compared to the calibration standard used by Waters on a Synapt G2-Si and Cyclic IMS. Our data shows that polyalanine standards contain a mixture of isomers with varying mobilities, making them unsuitable for IMS calibration.
In a third project, we developed an LC-MS/MS method, using UPLC and a triple quadrupole in multi-reaction monitoring mode, to investigate the activities of nitrilase enzymes on different substrates under different reaction conditions. Known and newly identified extremophile nitrilases were incubated with a large set of substrates and under a variety of reaction conditions to study substrate promiscuity and activity in harsh reaction conditions (acidic/basic pH, organic solvents and elevated temperature). Our data show that the tested nitrilases differ not only in their substrate specificity, but also in regard to performance under adverse reaction conditions.