Masaru Miyagi, Ph.D.

Faculty Appointments Center for Proteomics and Bioinformatics: Assistant Professor Department of Pharmacology: Assistant Professor (Secondary) Department of Ophthalmology: Assistant Professor (Secondary)

Academic History

Positions and Employment

Research Interests

Development of Analytical Methods

We are motivated to develop analytical methods for proteome profiling and protein structural characterization. Mass spectrometry is the pre-eminent technology in analytical protein chemistry. However, current methods on the front end of mass spectrometry analysis are not always taking advantage of modern mass spectrometry technologies.Development of advanced analytical methods on the front end of mass spectrometry analysis is therefore important in advancing the study of proteins. The following methods have been developed or are being developed in our laboratory.

Proteolytic ¹⁸O labeling

The proteolytic ¹⁸O labeling method determines the relative ratios of individual proteins between two samples. This technique utilizes a protease and water (H₂¹⁶O and H₂¹⁸O) to produce labeled peptides; peptides in one sample are labeled with ¹⁶O, and peptides in the other sample with ¹⁸O. Both samples are mixed in a 1:1 ratio and subjected to mass spectrometric analysis to identify and quantify the proteins from which the peptides originated.Our laboratory has found highly efficient oxygen labeling conditions for Lys-N, Lys-C, porcine trypsin and Streptomyces erythreus trypsin.Our current efforts are focused on establishing an experimental workflow that can be done entirely in a single tube in order to eliminate the peptide loss during the labeling procedure.

His-HDX-MS

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method also provides the pK_a values of histidine imidazole rings by monitoring the HDX rates as a function of pH.This method is useful to study the changes of protein conformations induced by ligand binding, protein-protein interactions, etc.We are currently studying what chemical and physical factors influence the t_1/2 of HDX and pK_a values. qThe elaboration of His-HDX mechanisms will provide a firm foundation for correctly interpreting the results obtained using this method, and will ultimately allow us to precisely predict changes in the microenvironment of histidine residues from changes in the t_1/2 and pK_a values.

Characterization of Proteomes

We use mostly the proteolytic ¹⁸O labeling method to quantitatively profile proteomes (including protein expressions, post-translational modifications and protein-protein interactions) of cells/tissues/organs to systematically study the physiological changes occur in a particular disease or with external stimuli.Such proteomic studies provide a .snapshot. of proteomes under defined conditions. This knowledge is essential in understanding the pathophysiology of a disease and providing a foundation for the development of novel therapeutic strategies for the disease.

The major focus of proteomics research in our laboratory is to elucidate what biological processes are dysregulated in diabetic retinopathy (a major complication of diabetes and the leading cause of new cases of blindness among adults 20-74 years old), and to find ways to restore the dysregulated processes. Other efforts in our laboratory include proteome profiling of 1) diabetic kidney, 2) diabetic bladder, 3) C. elegans, 4) platelet-derived microparticles, and 5) osteoarthritis/rheumatoid arthritis synovial fluid.

Characterization of Protein Conformational Changes

We use His-HDX-MS to study the conformational changes of proteins induced by ligand binding, protein-protein interactions, etc.Histidine residues are useful intrinsic probes for investigating structural changes in proteins for two primary reasons. First, the imidazole C₂-hydrogen exchanges with deuterium in the solvent D₂O; this process is a sensitive measure of solvent accessibility.Second, the acid dissociation constant (pK_a) of a histidine imidazole group changes in response to the adjacent ionizable group(s), providing an indicator of the electrostatic environment of the histidine residue.The following projects are being conducted in our laboratory.

1) ligand-induced changes in E. coli dihydrofolate reductase (DHFR)

2) changes in rhodopsin structure upon photoactivation and G protein (transducin) binding

3) changes in anthrax protective antigen structure upon binding to a receptor protein

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