GBMSDG Meeting Archives
April 13, 2010
Professor Brandon Ruotolo
University of Michigan
Applying Ion Mobility-Mass Spectrometry to Frontier Challenges in Structural Biology
1 Kendall Square, Cambridge
Special thanks to Waters for sponsoring this event. It is the active participation of our sponsors that allows us to keep the fees for membership, dinners, drinks, speakers, etc. so low.
Structural biology is ultimately concerned with determining high-resolution structures of all the functional macromolecules within living cells and tissues. While high-detail structural information can be obtained, for example, by X-ray diffraction analysis, this experiment requires the availability of a sufficient quantity of homogenous material and definition of suitable crystallization parameters. Both conditions are often difficult to meet, and thus the number of structures of multi-subunit complexes deposited in structural databases remains relatively low. Alternative methodologies such as electron microscopy (EM) and small angle X-ray scattering (SAXS) allow the determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited to homogeneous complexes. Consequently, there is a need to develop new approaches that define the subunit stoichiometry, composition, and shape of heterogeneous macromolecular complexes of biological importance. Over the past several years, we have been developing ion mobility-mass spectrometry (IM-MS) methods for the analysis of large protein assemblies. IM separates ions based on their ability to traverse a chamber filled with inert neutral molecules under the influence of a weak electric field. Ions that are large undergo a greater number of collisions with neutral molecules and thus take more time to elute from the chamber than smaller, more compact molecules. Ion size is, therefore, the primary information content of IM separation and computational approaches can be used in conjunction with this information and MS data to assign the overall topology and structure to large multi protein complexes. Alternatively, energetic collisions can be used to activate and unfold the complex in the gas-phase. This approach, termed collision induced unfolding (CIU), is unique to IM-MS and can provide localized structural information on multi-protein-ligand binding sites from complex mixtures. This presentation will focus on recent illustrative examples where the IM-MS approach is applied to resolve the structural features of disease-associated protein complex systems.
About Brandon Ruotolo
Brandon T. Ruotolo received his B.S. degree in Chemistry from St. Louis University (St. Louis, MO, USA.) in 1999. While there, he was able to participate in some undergraduate research under the direction of Dana M. Spence (currently at Michigan State, Department of Chemistry) in the fields of capillary-based flow injection and enzyme reaction kinetics. He then went on to join David H. Russell’s research group at Texas A&M University (College Station, TX, USA.). While at A&M, Brandon was primarily involved with the design, development, and implementation of hybrid Ion Mobility-Mass Spectrometry (IM-MS) instrumentation. During the course of his Ph. D. work, Brandon charted the peak capacity of IM-MS separation, performed proof-of-concept studies on the utility of IM-MS for phosphopeptide screening, investigated the utility of the method for analyzing short DNA segments, aided in the implementation of tandem MS instrumentation on several IM-MS instrument platforms, and performed several studies aimed at determining the gas-phase conformation(s) of biologically relevant peptides and protein segments. These instrument and method development projects were carried out in collaboration with both Ionwerks Inc. (Houston, TX., USA.), and the NIH (Baltimore, MD., USA.). Brandon received his Ph. D. in 2004. Directly after his graduate work, Brandon joined Prof. Carol Robinson’s laboratory at the University of Cambridge in the United Kingdom. During his time there, Brandon spearheaded the development of IM-MS for the analysis of multi-protein complex structure. His work led to the initial fundamental observations on the stability of protein quaternary structure in the absence of bulk solvent, elucidated protein-small molecule interactions, and produced a more-refined mechanistic picture for Protein-Protein dissociation reactions in the gas-phase. Instrument development work for these and other projects were carried out in close collaboration with Waters Corporation Research and Development (Manchester, UK), including a significant role in the development of both generations of the Synapt IM-MS instrument platforms – the first commercial mass spectrometry system to incorporate ion mobility separation. During his final year in the UK, Brandon was made the Waters Research Fellow at the University of Cambridge – a research position created for Ruotolo by Waters Corp to carry out independent research. In 2009, Brandon was appointed as an Assistant Professor in the Department of Chemistry, at the University of Michigan. Brandon’s current research interests continue to revolve around the development of techniques and technologies aimed at better-understanding protein structure. Specifically, Brandon continues to explore the utility of IM separation for the study of biological complexes, tandem mass spectrometry as a tool for elucidating the structure of protein complexes, and the fundamental principles of electrospray ionization. Collaborators for these projects include individuals at: Waters Ltd. (Manchester, UK.), University of Cambridge Departments of Biochemistry and Chemistry (Cambridge, UK.), the University of California, (Santa Barbara and Davis CA., USA.), Texas A&M University (College Station, TX USA.), and Lawrence Livermore National Laboratory (Livermore CA., USA).