Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• Current activity-based protein profiling methods rely on irreversible covalent modification, limiting probe applicability.
• Enzymes lacking covalently bound substrate intermediates are challenging to profile using existing techniques.

Purpose of the Study:

• To develop and validate a complementary, affinity-based labeling approach for profiling metalloproteases.
• To enable the characterization of enzymes that do not possess covalently bound substrate intermediates.

Main Methods:

• Design of affinity-based probes incorporating a peptidyl hydroxamate zinc-binding group, a fluorescent reporter, and a photolabile diazirine.
• Photolysis of the probe to generate a covalent linkage between the probe and the target metalloprotease.
• Analysis of labeled proteins via SDS-PAGE to distinguish target enzymes.

Main Results:

• The affinity-based approach selectively labeled metalloproteases in complex protein mixtures.
• Labeling efficiency was dependent on enzyme catalytic activity, with inhibitors proportionally reducing labeling.
• Unique enzyme "fingerprint" profiles were generated using probes with varying P(1) amino acids, aiding substrate specificity determination.
• Successful demonstration of large-scale metalloprotease profiling in yeast.

Conclusions:

• The developed affinity-based labeling method is a viable alternative to activity-based profiling for metalloproteases.
• This approach facilitates selective enzyme identification, characterization, and substrate specificity determination.
• The method holds potential for in situ screening of metalloprotease inhibitors and large-scale proteomic studies.