Development of mass spectrometry and ion mobility methods and instrumentation for the characterization of proteins from primary sequence to higher order structure

The development of methodology and instrumentation associated with mass spectrometry (MS) and ion mobility (IM) spectrometry for the characterization of proteins and other complex biomolecules was described and evaluated. 193 nm ultraviolet photodissociation (UVPD), when applied in both middle-down and top-down proteomic workflows, produced higher sequence coverage when compared to collisional activation methods. Using carbamylation of lysine to restrict tryptic cleavage to less common arginine residues, larger peptides were produced that benefited from the enhanced sequence coverage of UVPD to enable better characterization of protein sequence regions typically missed in traditional bottom-up workflows. When UVPD was combined with gas-phase charge reducing proton transfer reactions selectively applied to reduce spectral complexity, these gains were even more significant and enabled in-depth characterization of proteins as large as 56 kDa. MS and IM technologies were further applied to the characterization of higher order molecular structure. A novel method was developed to measure collision cross sections (CCS) of intact protein ions using the decay rate of the time domain transient signal produced by an Orbitrap mass analyzer. This method produced measurements with < 10% deviation from those generated by ion mobility without the need for specialized equipment or instrument modification. Methods to couple IM separations to Orbitrap mass spectrometry were developed and evaluated. The application of absorption mode processing to Fourier transform IM multiplexing techniques resulted in significant improvements in both resolution and signal to noise ratio compared to traditional magnitude mode processing. Performing UVPD on glycerophospholipids following IM separation enabled the accurate measurement of CCS from mixtures of double-bond isomers even when they cannot be fully resolved by standard IM instrumentation. Finally, a reduced pressure IM drift tube was designed and built to interface with a UVPD equipped Orbitrap mass spectrometer and used to characterize heterogeneous higher-order gas-phase protein structures using post IM UVPD.