The structures assumed by biological molecules as they perform their specific functions, as well as what changes cease those functions, have been of great interest to biologists and chemists alike. Many methods exist for probing the structures of these molecules in solution, each with their strengths and weaknesses. Techniques for ionizing proteins and large (>2 MDa) complexes, even from biologically relevant solutions, have introduced mass spectrometry as a means of probing the structures of these molecules in the gas phase. Advantages of mass spectrometry include its greater speed and sensitivity, as well as its ability to examine heterogeneous mixtures and complexes. However, difficulties arise in relating gas phase structures to those observed in solution. The proposed work is focused at better understanding how the transition from solution to gas phase influences the structures of biological macromolecules, specifically, what memory ions that are currently indistinguishable by many gas phase techniques might have of their solvent. This will be achieved by coupling a high resolution (R~100) static-field IMS drift tube, capable of measuring absolute collision cross sections, with mass spectrometry and other characterization techniques, most notably electron capture dissociation (ECD). ECD has become a powerful tool in protein sequencing, probing secondary structure, and identifying sites of post-translational modifications, but the vast majority of studies have examined a charge state of a biomolecule as a whole. IMS has shown that multiple conformations coexist across a charge state of a protein, and that solvent directly influences the distribution of conformations;little is known regarding how differences in structure affect either ECD fragmentation pathways or efficiencies. To this end, one aim of this study is to better understand how the cross section of an ion affects both the capture and fragmentation efficiencies of ECD. Performing this experiment requires building an IMS drift cell that can couple easily to several mass spectrometers. While ECD experiments must be performed in the FT/ICR cell, the timescale of the mass analysis is not amenable to obtaining mass-to-charge values for all ions as they exit the drift tube;instead, nested measurements will be made using IMS coupled to a quadropole (Q-) TOF instrument. The IMS-Q-TOF can also be employed for determining collision cross sections for ions too large to be analyzed by FT/ICR. Currently, the only collision cross sections for large (100 kDa and greater) complexes have been measured with the travelling wave IMS, a low-resolution (R~20) technique that only provides relative collision cross sections. When coupled to the FT/ICR, the drift tube will facilitate the measurement of ECD spectra of mobility selected ions for which the absolute collision cross sections are known. The process will ultimately improve knowledge of the conformations ions exhibit in vacuum, how solvent influences those conformers, and how to potentially employ this knowledge in both structural analysis and biomolecular sequencing.
The gas phase provides an interesting regime in which to probe biological macromolecules and complexes too large and/or complicated to be probed by solution phase methods. Relationships between the gas phase conformations of these molecules and the solvent from which they came could provide complimentary information to that obtained with methods such as nuclear magnetic resonance and X-ray crystallography. To this end, ion mobility spectrometry will be employed to obtain absolute cross sectional measurements of these molecules, as well as to select specific conformational subsets for characterization with mass spectrometric techniques, initially electron capture dissociation, with a focus on biologically relevant systems, including (but not limited to) the Barnase/Barstar complex, the sigma 54 (Ntr-C4-RC) complex, and the anthrax toxin complex.
|Merenbloom, Samuel I; Flick, Tawnya G; Williams, Evan R (2012) How hot are your ions in TWAVE ion mobility spectrometry? J Am Soc Mass Spectrom 23:553-62|
|Flick, Tawnya G; Merenbloom, Samuel I; Williams, Evan R (2011) A simple and robust method for determining the number of basic sites in peptides and proteins using electrospray ionization mass spectrometry. Anal Chem 83:2210-4|
|Merenbloom, Samuel I; Flick, Tawnya G; Daly, Michael P et al. (2011) Effects of select anions from the Hofmeister series on the gas-phase conformations of protein ions measured with traveling-wave ion mobility spectrometry/mass spectrometry. J Am Soc Mass Spectrom 22:1978-90|
|Flick, Tawnya G; Merenbloom, Samuel I; Williams, Evan R (2011) Anion effects on sodium ion and acid molecule adduction to protein ions in electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 22:1968-77|