Located in Ann Arbor, Michigan, USA, the University of Michigan is one of the world's top research institutions. The research team of Brandon Ruotolo, a professor of chemistry at the University of Michigan, is focused on developing new tools and methods to determine the three-dimensional (3D) structure and stability of proteins and multi-protein assemblies important in biology and medicine. The team used ion mobility-mass spectrometry (IM-MS) to develop innovative methods, combined with other structural biology tools, to solve some of the truly challenging problems in the field of structural genomics.
Structural Genomics Research Group, University of Michigan
The long-term goal of the project team is to use IM-MS data with other protein structure assays to support the emerging field of structural biology, combining multiple data from different techniques to generate more complete protein assembly data than using any single tool. Labs were purchased to help the team continue to make breakthroughs in the field.
Dr. Ruotolo explains: "Our work focuses on the intersection between structural biology and drug discovery research. Waters SELECT SERIES Cyclic IMS allows us to conduct new experiments and push boundaries in almost every field.
Figure 1. During the development of the Waters SELECT SERIES Cyclic IMS, Dr. Ruotolo and his team worked closely with Waters R&D personnel.
New approaches to structural biology
The study of the association between protein structure and function is essential for understanding biochemistry and human disease. To understand the role of protein structure in function, and any changes that may occur in disease states, Ruotolo's team is working to explore linking these biophysical parameters/factors. Protein stability (often reported as the free energy of protein unfolding) is an important aspect that is important for understanding protein structure and function. However, one of the main bottlenecks is the limited analytical techniques for determining the structure of macromolecular protein complexes.
Ruotolo's team's research interests focus on multiple topics related to structural biology, such as developing IM-MS instruments and building computational tools for IM-MS data analysis, and generating 3D models. There are also projects focusing on Collision Induced Unfolding (CIU) of protein complexes in the IM-MS gas phase, where researchers are establishing a technique for protein inhibitor discovery and rapid biotherapeutic characterization; There are also studies involving protein self-assembly, aggregation, and amyloid formation.
Dr. Ruotolo's work focuses on IM-MS as an emerging technique to determine the composition, size, and topological organization of protein assemblies using a small number of samples in the presence of impurities and structural heterogeneity. During the development of the Waters SELECT SERIES Cyclic IMS, Dr. Ruotolo's research team worked with Waters R&D staff to develop structural biology through IM-MS technology and push the boundaries of scientific discovery as a long-term goal.
Figure 2. The Ruotolo research team uses the SELECT SERIES Cyclic IMS instrument to solve challenges in the field of structural genomics.
Functions of ion mobility mass spectrometry (IM-MS).
Rapid characterization of protein 3D structures and the resulting polymers remains one of the great challenges facing modern biological and medical sciences. Especially in the drug discovery process, IM-MS technology is playing an increasing role in characterizing these functional complexes.
When ion mobility (IM) is coupled with MS, isomers can be separated based on shape and m/z, and clearer mass spectrometry data and accurate collision cross-section (CCS) values can be provided. Collision dissociation also helps scientists break down proteins and record their amino acid sequences, and one of the goals of Ruotolo's team is to extend such sequencing efforts to very large protein complexes.
DR. BRANDON RUOTOLO, PROFESSOR OF CHEMISTRY AT THE UNIVERSITY OF MICHIGAN, SAID: "WE ARE LOOKING AT SOME COMPLEX PROTEIN COMPLEX MODELS TO SEE THE SEQUENCE COVERAGE WE OBTAINED. The results of using Waters SELECT SERIES Cyclic IMS look fantastic! ”
The Waters SELECT SERIES Cyclic IMS is engineered to achieve higher ion mobility resolution; Support for ion selection by mass and mobility, a feature exclusive to Cyclic IMS. These features of Cyclic IMS provide flexibility and powerful capabilities for cutting-edge research, enabling high-level researchers to unlock new scientific discoveries.
Dr. Ruotolo describes the instrument's impact in its research area: "The Waters SELECT SERIES Cyclic IMS is a very special device that can do all kinds of interesting work and influence some of the key questions in the field of biochemistry today, which is of great help to researchers. ”
Figure 3. During the development of Waters SELECT SERIES Cyclic IMS, Dr. Ruotolo worked with Waters to push the boundaries of scientific discovery.
One of the research directions of structural biology: CIU
One of Ruotolo's team's research projects is to explore the potential of Collision Induced Unfolding (CIU) as a fingerprinting technique that can be used to study the structure and stability of proteins, protein complexes, and protein ligand complexes.
Collision activation of protein assemblies typically results in a large number of partially folded intermediates that are stable over the millisecond time frame and can provide a range of information relevant to the structure of isolated protein complexes. Dr. Ruotolo explained the study: "Our team is probably best known for their work using the CIU, which we use to measure the stability of proteins. These are key attributes that can be used to discover new biotherapeutics, or to obtain biophysical information about how proteins target or interact with drugs. Calorimetry is currently the benchmark technology in the pharmaceutical industry, but it is very slow. It requires a relatively large number of samples and usually requires purified samples to perform. The CIU is very fast, and when running CIU experiments with instruments in our lab, each sample takes only a few seconds. We are using this technology to work with numerous pharmaceutical companies to understand how biotherapeutics are discovered, or how different biotherapeutics alter the proteins involved. The technology is in the development phase, and the unique strengths of Waters SELECT SERIES Cyclic IMS are ideally suited to the job. ”
Ruotolo's team's work also seeks to capture the characteristics of biotherapeutic drug unfolding and link them to therapeutic-related structural differences, ultimately constructing high-throughput methods for rapidly analyzing novel therapeutic proteins. Compared to many common screening techniques, CIU trials are highly informative, enabling more targeted drug screening and improved protein engineering methods.
DR. BRANDON RUOTOLO, PROFESSOR OF CHEMISTRY AT THE UNIVERSITY OF MICHIGAN, SAID: "THERE IS NOW A HUGE NEED FOR QUANTITATIVE ANALYSIS OF DIFFERENT BIOMARKERS OF NEURODEGENERATIVE DISEASES BECAUSE WE NEED METHODS TO DIAGNOSE PATIENTS EARLY. However, because biomarkers are structural in nature, it is difficult to establish analytical methods around these types of targets to solve this problem. We are using the Waters SELECT SERIES Cyclic IMS and its CIU capabilities to build an analytical method based not only on the composition or properties of a biomolecule, but also on its structural state. The analytical methods we are building to solve this problem are called CIU2, and these methods creatively leverage the capabilities of the SELECT SERIES Cyclic IMS system. ”
Figure 4. The Ruotolo team leveraged the capabilities of the Waters SELECT SERIES Cyclic IMS instrument to explore the potential of different CIU approaches.
Other items include the role of protein misfolding and aggregation in disease. Most protein molecules must fold into specific 3D structures to be functionally active.
Looking to the future
The two focus areas of the biomass spectrometry facility are the analysis of intact proteins, multi-protein complexes, and protein structures; and combined with quantitative analysis to identify and purify compounds from complex mixtures. Researchers at the University of Michigan have a keen interest in liquid phase separation and natural product analysis using the Waters SELECT SERIES Cyclic IMS instrument. Dr. Ruotolo sees this new facility and instrument as a resource for university researchers to continue pushing boundaries across multiple scientific fields: "Any research in the field of biochemical isomers is a hot potential application for Waters SELECT SERIES Cyclic IMS. It's great to have this facility because we need enough bandwidth to help different researchers on campus. That's my ultimate motivation as a scientist, which is to make sure that we can use these measurements to help people and really bring them real benefits. This is one of the most fascinating aspects of measurement science, where you can develop some results in the lab, such as new techniques for measuring biomarkers. The real joy is not developing this technology for myself, but seeing it put into practical use and helping people explore. I would like to see this facility and Waters SELECT SERIES Cyclic IMS become a great resource to help people do the scientific work they want to do. ”