Novel methods for manipulating ion types in the solution and gas phases for the structural analysis of biomolecules using Mass Spectrometry

Mass Spectrometry has become a valuable tool for the analysis of a variety of molecules, making it applicable to many fields. The advent of nanoelectrospray ionization (nESI) as a soft/low energy ionization technique has enabled the analysis of large, intact biomolecules. Most mass spectrometry experiments consist of three main steps: ionization, probe step(s), and mass analysis. The present work focuses on a variety of methods for altering ion types at various stages of the mass spectrometry experiment to affect ion fragmentation. Ion types can be manipulated in the solution/droplet phases using novel nESI emitters, generated from borosilicate theta capillaries. These nESI emitters enable the mixing of two solutions as they are sprayed into the mass spectrometer. This technique has been used to manipulate protein charge states (i.e. protein folding and unfolding) and to demonstrate peptide/protein analyte-reagent complex formation and covalent modification. This technique provides a simple and inexpensive method for manipulating ion types as the ions are generated during the electrospray process on the sub-millisecond timescale. These nESI emitters are also expanded to longer solution mixing times through the use of electroosmotic flow (EOF) between the two channels of the theta capillary prior to mass analysis. This work presents initial efforts to use theta capillaries to develop a lab-in-a-tip to provide for the manipulation of ion types on short timescales just prior to mass analysis. Additionally, ion types can be manipulated once the ions are in the gas-phase and trapped inside the mass spectrometer via ion/ion reactions. This work presents ion/ion reactions with reagents containing chromophores, which can be activated via ultraviolet photodissociation (UVPD) to generate radical peptide cations. Altering ion types in this way provides complementary sequence information upon collision induced dissociation (CID) when compared to CID of the even electron species. The McLuckey group is well known for work with ion/ion reactions to modify ion types and to conjugate biomolecules through covalent chemistry in the gas-phase. However, the kinetics and energetics of these reactions are not well known. This work will provide a method for measuring ion/ion reaction kinetics using dipolar DC CID (DDC-CID), which was previously developed in the McLuckey lab. Knowledge of gas-phase ion/ion reaction kinetics and energetics will provide insights for improving current ion/ion reaction efficiencies as well as for improving reagent design for future ion/ion reactions