DEVELOPMENT AND CHARACTERIZATION OF ELECTROSPRAY IONIZATION MODES IN MASS SPECTROMETRY: APPLICATIONS IN PROTEIN ANALYSIS AND BEYOND

Electrospray ionization (ESI) has emerged as a widely used soft ionization method, facilitating the introduction of intact molecular ions into mass spectrometers for diverse applications. Despite its popularity, ESI in its current form still faces certain non-ideal limitations. One ongoing research direction is focused on reducing the flow rate for ESI, a crucial parameter that correlates with the size of initial charged droplets generated. Smaller initial charged droplets have exhibited improved performances, including enhanced ionization efficiency, reduced nonspecific adducts, and minimized sample consumption. It would be of significant interest to measure and investigate ultra-low ESI flow rates for downscaled study.This dissertation presents a femto flow electrospray ionization method with flow rates ranging from 240 fL/min to the low picoliter per minute level. It was conducted and measured using a submicron emitter tip and relay ESI configuration. Signature analyte ion current intensities and profiles were observed.Furthermore, the development of femto electrospray ionization, which generates ultra-low ionization currents in the range of 10s fA to 100s fA using a conventional micrometer-sized emitter for nanoESI, is reported. To the best of our knowledge, this represents the lowest reported value. Notably, the mass spectrometer response revealed that femtoESI exhibits the charge state manipulation of analytes, especially evident for protein analysis, where proteins tend to be preserved in their native charge states.Given charge states of protein ions are essential to grasp the behavior of proteins, such as their conformations, dissociation mechanisms, transmission in solution and in the gas phase, evaluation of femto electrospray performance on protein analysis was conducted by utilizing supercharging agents. This investigation shed further light on the mechanisms of protein supercharging and ionization behavior. Mass spectra obtained by on-demand alternation between femtoESI and nanoESI indicated that femtoESI enabled better desolvation and preservation of native protein structures.The distinctive characteristics of femtoESI, in comparison to nanoESI, offer profound insights into protein analysis and the underlying mechanisms of electrospray ionization, pushing the boundaries of ESI parameters to new frontiers.