Presenter Information

Scott Coffin, University of Wyoming

Department

Department of Chemistry

First Advisor

Dr. Franco Basile

Description

Enzymes such as Trypsin are used extensively to study proteins (i.e. proteomics) due to the simplicity of use, and the enzyme’s ability to cleave proteins specifically at Arginine (R) and Lysine (K), which allows the fragments to be easily analyzed using t andem Mass Spectrometry (MS/MS) measurements. However, enzymes have a limited useful shelf life, must be stored at - 80 ° C, requires many manual procedural steps, and most importantly it requires an incubation time of 7 - 8 hours (overnight), thus making its implementation time - consuming and unsuitable for automated operation. In contrast to this commonly used technique, a new protocol which utilizes two techniques, including the reagent - less microwave cleavage at aspartic acid (D) and the microwave - assisted c leavage of disulfide bonds using a reducing agent (dithiothreitol, DTT) allows for proteins to be cleaved and analyzed by researchers in as little as five minutes, and in a flow - through manner, allowing automation.. The goal of this project was to optimiz e the microwave radiation heating procedure to cleave disulfide bonds in proteins. Variables like heating temperature and time, DTT concentration and pH were all tested and conditions were found for the rapid and efficient cleavage of disulfide bonds in p roteins.

Comments

Oral Presentation, UW Honors Program, Wyoming NSF EPSCOR

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Optimization of Non - Enzymatic Microwave Cleavage at Aspartic Acid and Disulfide Bons

Enzymes such as Trypsin are used extensively to study proteins (i.e. proteomics) due to the simplicity of use, and the enzyme’s ability to cleave proteins specifically at Arginine (R) and Lysine (K), which allows the fragments to be easily analyzed using t andem Mass Spectrometry (MS/MS) measurements. However, enzymes have a limited useful shelf life, must be stored at - 80 ° C, requires many manual procedural steps, and most importantly it requires an incubation time of 7 - 8 hours (overnight), thus making its implementation time - consuming and unsuitable for automated operation. In contrast to this commonly used technique, a new protocol which utilizes two techniques, including the reagent - less microwave cleavage at aspartic acid (D) and the microwave - assisted c leavage of disulfide bonds using a reducing agent (dithiothreitol, DTT) allows for proteins to be cleaved and analyzed by researchers in as little as five minutes, and in a flow - through manner, allowing automation.. The goal of this project was to optimiz e the microwave radiation heating procedure to cleave disulfide bonds in proteins. Variables like heating temperature and time, DTT concentration and pH were all tested and conditions were found for the rapid and efficient cleavage of disulfide bonds in p roteins.