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Characterization of irreversible inhibition of proteases by mass spectroscopy

Author

Zhonghua Walter Yu, University of the Pacific

Date of Award

1995

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Chemistry

First Advisor

Patrick R. Jones

First Committee Member

Silvio Rodriguez

Second Committee Member

Michael J. Minch

Third Committee Member

James W. Blankenship

Fourth Committee Member

Alma L. Burlingame

Abstract

Proteases are present in all living organisms and are involved in various biological processes. Inhibition of protease activities in disease-causing agents is one strategy for rational drug design. Characterization of the protease inhibition processes is essential for understanding the inhibition mechanisms and for developing efficient therapeutics. This work represents a major challenge in analytical biochemistry. In this study, a strategy based on mass spectrometry has been developed to characterize irreversible inhibition of proteases. Five proteases representing three of the four protease classes were irreversibly inhibited by various irreversible inhibitors, some of which are potential drug candidates. In all the cases, the stoichiometry of each of the protease/inhibitor complexes was determined by electrospray ionization mass spectrometry through measurement of the complex's molecular weight. The inhibited proteases were then enzymatically cleaved and the resulting peptides isolated for further characterization by high performance tandem mass spectrometry. Attention was focused on the determination of the site(s) of the modification and the reaction mechanisms involved. High energy collision induced dissociation mass spectra of each modified peptide provided information on the exact modification site(s) and the detailed chemical nature of the covalent complex. The serine protease trypsin, the cysteine protease cruzain, and the aspartic proteases, HIV-2 protease and SIV protease, were covalently modified only at one amino acid residue, while the aspartic protease, HIV-1 protease, was found to be modified at three sites by the haloperidol derivative compounds. In addition, mass spectrometry has been applied to characterize the plasma glycoprotein, biotinidase, and to obtain partial peptide sequences of a membrane-bound protein, UDP-GalNac:polypeptide N-acetylgalactosaminyl transferase, using a low picomole quantity of sample.

Pages

263

Recommended Citation

Yu, Zhonghua Walter. (1995). Characterization of irreversible inhibition of proteases by mass spectroscopy. University of the Pacific, Dissertation - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/2805