Influence of Ion Charges to the Theoretical Calculated Cross Section and the Resolving Power and Accuracy of the Experimental Cross Section in Trapped Ion Mobility Mass Spectrometry

The overarching goal of the work in the following doctoral thesis is to improve the capacity of trapped ion mobility mass spectrometry to contribute to our understanding of molecular structure. This involves the separation and analysis of conformationally related species in a complex mixture by use of ion mobility separations. The accuracy with which we can compute and measure cross section is explored deeply because a comparison between the theoretical and experimental cross section is essential to the characterization of molecular structure. We begin by giving an overview of the progress in structural biology: from the early years of X-ray imaging structures in the solid state to the recent digital reconstruction of molecular structures from solution or vitreous phase analysis. We present the transformative influence of technological innovations from physics, chemistry, and molecular biology to the field of structural biology. The structural biology community now recognizes that molecular conformational dynamics, where structures evolve in time and with biochemical interactions, governs many physiological processes and has yet been fully explored. The common challenge in relevant analytical and biophysical technologies is maintaining resolution when analyzing mixtures of conformers and accessing the dynamics of the biomolecular structure. We describe an emerging method, ion mobility-mass spectrometry (IM-MS), as a new prospective technology that simultaneously performs the dual task of separating species in mixtures based on their size and shape and probing information related to the structural conformation of each species. We focus on the recently introduced IM-MS platform of trapped ion mobility mass spectrometry (TIMS) as a promising technology for structural characterization as well as tracking structural changes of biomolecules in the gas phase. The structural characterization capacity of TIMS is based on the comparison between the theoretically calculated cross sections and the experimentally measured cross section. In chapter 2, we address the accuracy of the theoretical cross section calculation. Here, we systematically investigate the influence of the ion charge distributions on the momentum transfer cross section of analyte ions in the absence of any structural dynamics. To this end, we calculate momentum transfer cross sections for carbon cluster model systems and assessed how they change when varying temperature and the polarizability of the buffer gas as well as mass, total charge, and charge distribution of the ion. We find that the significance of the ion charge distribution increases with ion charge, buffer gas polarizability, and charge localization. Conversely, the significance of the ion charge distribution decreases with buffer gas temperature and molecular mass of the analyte ion. Our data indicates that accurate structural characterization on the basis of IMS-MS data measured in nitrogen buffer gas for ions similar or smaller in mass (~3 kDa) or momentum transfer cross sections (400 – 500 Å2) and even those as large as ~12kDa, depend significantly on the details of the charge distribution. Finally, our data indicate that the molecular conformation can influence the calculation of the momentum transfer cross section via forming clusters of charge density at certain structural folds. In chapter 3, we address the accuracy of the experimental cross section measurement. Here, we explore the influence of the entrance pressure and the reduction of the ramp rate to optimize the experimental resolving power as well as the accuracy of the measured ion mobility. Our results indicate that the experimental resolving power in TIMS at pressures of 2.52 and 2.80mbar are within 5% from the predicted theoretical resolving power. A comparable assessment between Michelmann et al. and our results indicate that while the previous experimental resolving power was 100 at 2.90 mbar and ~120 at 3.1 mbar for tuning mix ion with m/z 622.2, we are able to obtain experimental resolving power of 220 for [SDGRG+H]+ and [GRGDS+H]+ with m/z 491.22. Not only are we able to achieve twice the experimental resolving power obtained by DT-IMS (R~100), our experimental resolving power is also twice as high as any previous TIMS based report. With this level of resolving capability, we are able to achieve baseline resolved separation of the isomeric reverse peptide ions [SDGRG+H]+ and [GRGDS+H]+ which have a 0.7% mobility difference. Also, we determine that the average of the absolute mobility values from all three pressures 2.00, 2.52, and 2.80 mbar are 0.996 and 1.010. A comparison of the absolute ion mobilities determined by three approaches, (TIMS calibration approach, our in-house approach to determine ion mobility value directly from measured arrival time described in Appendix D, and DT-IMS approach) show that these values differ by less than 1.5%. The frequency of biomolecular conformational variants in the range of 2-5% difference in cross section compares favorably to the capacity of TIMS to resolve less than 1% differences in cross section. These findings show that TIMS is a versatile and high-throughput instrument for the ion mobility-based separation and structural characterization for conformers in a biological mixtures.