Detecting and identifying glycoside hydrolases using cyclophellitol-derived activity-based probes

Area of Science:

• Biochemistry
• Enzymology
• Proteomics

Background:

• Detecting active enzymes in complex protein mixtures typically requires specific activity assays and challenging separations.
• Broadly reactive activity-based probes (ABPs) provide an alternative strategy for identifying active enzymes, particularly glycoside hydrolases (GHs).

Purpose of the Study:

• To outline activity-based protein profiling (ABPP) methods for detecting and identifying active retaining glycoside hydrolases (GHs) from microbial sources.
• To detail sample preparation, enzyme labeling, and identification strategies using ABPP.

Main Methods:

• Utilized activity-based protein profiling (ABPP) techniques for sensitive and selective detection of active retaining glycoside hydrolases.
• Employed protein sample preparation from bacterial lysates and fungal secretomes.
• Incorporated enzyme labeling with fluorescent probes, affinity-based enrichment, and peptide sequencing with isobaric labeling for enzyme identification.

Main Results:

• Demonstrated the efficacy of ABPP in separating, detecting, and identifying active retaining glycoside hydrolases from microbial samples.
• Achieved high sensitivity and selectivity in enzyme detection and identification.

Conclusions:

• ABPP serves as a powerful and complementary approach to traditional activity assays for studying active retaining glycoside hydrolases.
• The outlined methods facilitate the comprehensive analysis of GHs in complex biological matrices.