Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates.
Anal Biochem
; 567: 45-50, 2019 02 15.
Article
en En
| MEDLINE
| ID: mdl-30528915
ABSTRACT
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 17â¯pmol (0.29⯵g) for Glp-Phe-Ala-AMC and 43â¯pmol (0.74⯵g) 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.
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Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
/
Proteasas de Cisteína
/
Colorantes Fluorescentes
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2019
Tipo del documento:
Article