Monitoring enzymatic hydrolyses of small peptides using electrohydrodynamic mass spectrometry

Author

Steven Walter Salvato

Date of Award

5-1995

Degree Type

Thesis

Degree Name

Master of Science

Major

Chemistry

Major Professor

Kelsey D. Cook

Abstract

This work involves the development and testing of a new sample introduction technique to allow enzymatic hydrolyses of peptides to be monitored in real time using Electrohydrodynamic Mass Spectrometry (EHMS). The hydrolyses were carried out in aqueous acetate buffer. Because EHMS requires an organic cosolvent, the aqueous reaction mixture was mixed with a thioglycerol (TG) solution in a low dead volume tee before entering the mass spectrometer. The enzyme used in this work was carboxypeptidase-Y because of its ability to cleave most amino acids. Hydrolyses were performed on two carbobenzyloxy-protected dipeptides, and initial velocities were determined at different substrate concentrations. Kinetics information, such as the Michaelis constant (K_m) and maximum rate of the reaction (V_max), was obtained from the mass spectrometric data for one of the protected dipeptides and compared with that obtained using a well established method: the ninhydrin method with UV-Visible detection. The results revealed that systematic errors were present in the EHMS experiments. Possible causes for the systematic errors are contamination of the reaction mixture by TG and/or imperfect temperature regulation of the reaction. A pentapeptide, leucine-enkephalin, was hydrolyzed and monitored for 24 hours by EHMS (continuously) and the ninhydrin method (at timed intervals). Rate constants for the release of the first two amino acids were determined from the EHMS data and compared with those obtained through deconvoluting the optical data. Again, systematic errors were found to be present in the MS experiments. The results of this study prove for the first time that biochemical reactions can be monitored continuously using EHMS. Despite the systematic errors present, this work showed that EHMS can serve as a useful qualitative probe.

Recommended Citation

Salvato, Steven Walter, "Monitoring enzymatic hydrolyses of small peptides using electrohydrodynamic mass spectrometry. " Master's Thesis, University of Tennessee, 1995.
https://trace.tennessee.edu/utk_gradthes/11263