The WU Biomedical Mass Spectrometry Resource has been developing and applying MS-based tools to fundamental biological research and translating these to clinical research questions for over 35 years. Washington University provides a synergistic environment in which the Resource can contribute to both local and national efforts in MS-based research focused on genes, proteins, lipids, and metabolites that impact human health. All laboratories within the Resource address integrated driving biomedical projects (DBPs) that propel technological research and development (TRDs) by sharing and leveraging MS instrumentation, personnel, expertise and skills towards a common mission: collaborative development and implementation of the MS-based tools required to advance biological research.
The Structural Biology Laboratory, led by Resource PI Dr. Michael Gross, is located on the Danforth Campus of WU and is focused on understanding the interaction of proteins and their ligands (e.g., other proteins, nucleic acids, lipids, metabolites). Proteins rarely act alone and protein complexes are often quite large (e.g., several megadaltons). It is essential to understand protein stoichiometry and structure in their native states in order to discover their biology and develop targeted therapies. Traditional structural methods such as X-ray crystallography or NMR often require more material than is available or are difficult to apply due to the heterogeneity of the sample. MS methods developed and employed at the Resource allow studies of the proteins and ligands in their native states, including membrane-bound proteins, and require little sample due to the sensitivity of modern MS instruments. The Structural Biology Laboratory develops and employs hydrogen deuterium exchange (HDX) and chemical footprinting in collaborations with several laboratories in the United States and Europe. These studies take advantage of the developments in bottom-up proteomics made over the last decade, including those that originated in the MS-based Proteomics Core Laboratory within the MS Resource.
HDX MS is well-established for probing solvent accessible structure. At the Resource, HDX MS has been adapted to study kinetics and ligand binding order (e.g., the binding of four calcium ions by calmodulin). In addition, Resource staff and students have extended HDX MS to more challenging systems that depend on pH (e.g., diphtheria toxin, work with Dr. Alexey Ladokhin at Kansas University Medical Center), ionic strength (e.g., SecA, a study with James Cole at the University of Connecticut), and temperature. Resource personnel have also applied HDX MS to advance our understanding of antibodies and their epitopes (e.g., in a collaboration with Pfizer). They are also working on coupling HDX with top-down MS. Staff scientist Don Rempel is pioneering modeling algorithms that can extract kinetic and thermodynamic information from the large datasets generated during HDX MS experiments.
Chemical footprinting is also an old technique, but the performance improvements in MS make it especially attractive for structural studies. While deuterium labeling is labile, most chemical modifications to proteins are stable. This MS Resource pioneered the fast photochemical labeling of proteins (FPOP) using laser-generated hydroxyl radicals to covalently modify solvent accessible side chains on proteins and complexes (e.g., Her2 dimerization studies with Dr. Ron Bose). FPOP can provide structural data on ~100 kDa systems and is now being extended to probing the epitopes of antibodies as well as time-resolved detail on the folding of a single protein (e.g., Barstar). FPOP can also be applied to lipid-protein interactions and work is underway to characterize these in model systems described and identified by our colleagues in the MS Resource Lipidomics Laboratory. Resource staff and students also employ FPOP for protein interaction studies and this has proved to be effective in very large systems (e.g., FMO antenna complexes in membranes, a study with Dr. Robert Blankenship).
Staff scientist Weidong Cui is using top-down MS on an FTICR to tease apart large complexes to get at their stoichiometry and to employ fragmentation methods to understand the structure of the subunits. The development of native spray techniques followed by new isolation methods have allowed the use of electron activation to learn about the flexibility of regions of proteins in complexes (e.g., B-factor correlation). The advent of top-down MS and native spray is relatively recent, but coupling these techniques with high resolution MS permits the study of proteins in intact membrane systems (e.g., in collaboration with Dr. Stephen Sligar at the University of Illinois). Drs. Cui and Fong-Fu Hsu have characterized sphingolipids using top-down MS (e.g., Hsu and Cui). Dr. Cui is also working with staff scientist Henry Rohrs to map out the patterns of phosphorylation in signaling proteins with multiple sites (e.g., serine cluster domains). These developments will allow structural studies of downstream kinases, identified in the MS Resource Proteomics Core Laboratory, that are dysregulated in cancer.