Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates
| Autor: | Elena A. Dvoryakova, Brenda Oppert, Ashraf Oukasha Abd El-latif, E. V. Klyachko, Irina Yu. Filippova, Yulia A. Smirnova, Marina V. Serebryakova, Elena N. Elpidina, Tatiana A. Semashko, Mikhail A. Belozersky, Yakov E. Dunaevsky |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
Biophysics
Peptide Mass spectrometry 01 natural sciences Biochemistry Substrate Specificity Matrix (chemical analysis) 03 medical and health sciences chemistry.chemical_compound Cysteine Proteases Animals Amino Acid Sequence Tenebrio Molecular Biology 030304 developmental biology Fluorescent Dyes Detection limit chemistry.chemical_classification 0303 health sciences Chromatography Chemistry 010401 analytical chemistry Cell Biology 0104 chemical sciences Electrophoresis Matrix-assisted laser desorption/ionization Papain Larva Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Sequence Alignment Cysteine |
| Zdroj: | Analytical biochemistry. 567 |
| ISSN: | 1096-0309 |
| Popis: | A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). 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 matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect