Development of Ion Mobility-Mass Spectrometry as a High-throughput Approach for Structural Genomics.

Ion Mobility-Mass Spectrometry;Protein Complexes;Protein Structures;High-throughput;Chemistry;Science;Chemistry

Zhong, YueyangBrooks Iii, Charles L. ;

University of Michigan

关键词: Ion Mobility-Mass Spectrometry;    Protein Complexes;    Protein Structures;    High-throughput;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/107240/zhongyy_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Ion mobility-mass spectrometry measurement of proteins and multi-protein complexes is a tool of rapidly-growing importance in structural biology.However, many challenges remain in its development, including: optimizing protein size/shape measurements, developing methods for protein-protein interface characterization, and constructing high-throughput platforms for multiprotein topology determination. This thesis focuses on these challenges by developing new methods for ion mobility-mass spectrometry protein structure characterization.First, the performance characteristics of a second-generation travelling-wave ion mobility separator are assessed, focusing on those parameters that lead to the collection of high-accuracy, high-precision measurements of protein size. The conditions for high accuracy protein size measurements are significantly different from those optimized for separation resolution, indicating that a balance between these two metrics must be attained for traveling wave ion mobility separations of biomolecules.Second, in order to enable the high-throughput structural analysis of protein complexes and their subcomplexes, ion mobility-mass spectrometry is coupled with automated robotic sampling of carefully-titrated solution conditions. By altering solution ionic strength in concert with dimethyl sulfoxide content, the data collected shows that simple two-dimensional solvent screens are sufficient to disrupt protein-protein interfaces for a broad array of complex structures and folds.Ion mobility measurements captured for both intact assemblies and subcomplexes matched expected values from available X-ray data in all cases save two, where extreme disruption conditions were employed. Strong correlations between the disruption agents and chemical nature of interfacial interactions are observed.A key challenge for protein ion mobility measurements of intact proteins is accessing local, domain-level structure information.In a third set of experiments, gas-phase protein unfolding data were acquired for a range of monomeric proteins. The unfolding of multi-domain proteins, using either collision-induced unfolding or Coulomb-associated stretching, revealed a strong, positive correlation with known protein domain structures in solution.In a final set of experiments, the protocols developed here using model protein systems are applied to two multiprotein complexes of currently unknown structure: the heme oxygenase-2/cytochrome P450 reductase dimer and the human mitochondrial ribonuclease P hetero-hexamer. In these cases, IM-MS aided by elucidating either the chemical nature of the protein contacts or coarse-grained topologies of the complexes studied.