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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Stored Product Insect and Engineering Research » Research » Publications at this Location » Publication #355416

Research Project: Sustainable Management Strategies for Stored-Product Insects

Location: Stored Product Insect and Engineering Research

Title: Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates

item ELPIDINA, ELENA - Moscow State University
item SEMASHKO, TATIANA - Moscow State University
item SMIRNOVA, YULIA - Moscow State University
item DVORYAKOVA, ELENAA - Moscow State University
item DUNAEVSKY, YAKOV - Moscow State University
item BELOZERSKY, MIKHAIL - Moscow State University
item SEREBRYAKOVA, MARIANA - Moscow State University
item KLYACHKO, ELENA - Russian Academy Of Sciences
item ABD EL-LATIF, ASHRAF - Sohag University
item Oppert, Brenda
item FILIPPOVA, IRINA - Moscow State University

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2018
Publication Date: 2/15/2019
Citation: Elpidina, E.N., Semashko, T.A., Smirnova, Y.A., Dvoryakova, E., Dunaevsky, Y.E., Belozersky, M.A., Serebryakova, M.V., Klyachko, E.V., Abd El-latif, A.O., Oppert, B.S., Filippova, I.Y. 2019. Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates. Analytical Biochemistry. 567:45-50.

Interpretive Summary: Enzymes called cysteine peptidases are important in biological systems, including insects. However, these enzymes are difficult to study because they are unstable, so methods are needed to rapidly analyze their activity. We synthesized two new substrates that fluoresce when they are cleaved by these enzymes, so that we can now identify them when they are separated under a procedure called native PAGE. Once we find the activity, the enzymes can be identified through mass spectrometry. We demonstrated that this procedure can be used to identify major digestive enzymes in the gut of yellow mealworm larvae. Therefore, this technique is applicable to the study of these important enzymes in complex biological systems.

Technical Abstract: A method is described for the direct detection of cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, Glp-Phe-Ala-AMC (pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride) and Glp-Phe-Ala-AFC (pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride). The detection limit of the model enzyme papain was 17 pmol (0.29 µg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 µg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by MALDI-TOF and MS analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of cysteine peptidases in multi-component biological samples.