Most proteins do not exist by themselves in living systems but interact with other proteins in assemblies much as do members of a family. Mass spectrometry is becoming an important approach for investigating large protein assemblies in the gas phase. The goal of the research is to assemble and evaluate instrumentation for investigating macromolecular assemblies of proteins, to identify what proteins and how many constitute an assembly, to learn how they interface one to another, to discover what regions are free and what regions are part of the interface, and in the gas phase. The instrument is a Fourier transform ion cyclotron resonance mass spectrometer, and it will be built around a presently existing 7-tesla superconducting magnet. Once the instrument is built, protein assemblies will be introduced via a very gentle process called native electrospray ionization, and their masses measured with the highest performance instrument type that is available in mass spectrometry. Energy will then be added to the complex by high energy collisions to reveal the organization and interfaces of the assemblies. Results will be published in the scientific literature in widely disseminated journals and posted as abstracts and reports on a web site: http://msr.dom.wustl.edu/.
The study of proteins and their assemblies is central to solving problems in disease, understanding life, and perhaps even creating new sources of energy. We are motivated by this need to develop methods to study proteins, how they react and interact, and how they bind to other substances. To this end, we proposed developing high-energy collisional activation of protein ions, stored in the "trap" of a Fourier transform mass spectrometer, a powerful instrument for chemical analysis. Although this approach was too difficult to execute in the period of the grant, we were able to apply other methods to protein ions stored in this trap. One approach is to allow the proteins to capture electrons ("electron-capture dissociation" or ECD), causing them to fragment in ways that inform us of their nature. ECD of large proteins and protein complexes (assemblies of proteins) reveals new and unexpected information. For protein complexes, the sensitive complex holds together while various regions of the protein constituents break apart in smaller units, revealing their nature and their flexibility. This ability to fragment can also be used for large protein drugs, antibodies, to assist in their analysis. Protein drugs are the future in medicine. Finally, the means of introcucing proteins into the Fourier transform mass spectrometer can be used successfully to analyze lipid Nanodisks, informing us of their molecular weight, the number of lipids contained in these interesting disks, and the purity (dispersity) of the disks. This development sets the stage for use of lipid nanodisks for membrane-bound proteins, one of the "hold grails" of modern biological science.
