Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Profiling enzyme activities in vivo using click chemistry methods.

Anna E Speers1, Benjamin F Cravatt

  • 1The Skaggs Institute for Chemical Biology, Departments of Chemistry and Cell Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Chemistry & Biology
|May 5, 2004
PubMed
Summary

This study optimizes click chemistry for activity-based protein profiling (ABPP) in living systems. Optimized conditions reveal differences in enzyme activity between live and homogenized cancer cells, highlighting in vivo enzyme function.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

iScience·2021
Same author

Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug.

Cell reports·2021
Same author

Chemical proteomic analysis of palmostatin beta-lactone analogs that affect N-Ras palmitoylation.

Bioorganic & medicinal chemistry letters·2021
Same author

Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells.

Proceedings of the National Academy of Sciences of the United States of America·2021
Same author

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling.

Cell chemical biology·2021
Same author

Potentiation of amyloid beta phagocytosis and amelioration of synaptic dysfunction upon FAAH deletion in a mouse model of Alzheimer's disease.

Journal of neuroinflammation·2021

Area of Science:

  • Biochemistry and Molecular Biology
  • Chemical Biology
  • Proteomics

Background:

  • Understanding enzyme activity in vivo is crucial for deciphering physiological and pathological roles.
  • Existing methods for in vivo enzyme activity profiling are limited.
  • Activity-based protein profiling (ABPP) offers a way to study enzyme function.
  • Copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) is a powerful tool for bioorthogonal labeling.

Purpose of the Study:

  • To optimize reaction parameters for click chemistry-based ABPP.
  • To enhance the speed, sensitivity, and bioorthogonality of this profiling technique.
  • To compare enzyme activity profiles in living versus homogenized breast cancer cells.

Main Methods:

  • Detailed characterization of reaction parameters influencing click chemistry-based ABPP.

Related Experiment Videos

  • Application of optimized click chemistry conditions for in situ enzyme activity profiling.
  • Comparative analysis of enzyme activity in live and homogenized breast cancer cells using activity-based probes.

    • Main Results:

    • Identified optimal conditions for maximizing speed, sensitivity, and bioorthogonality in click chemistry-based ABPP.
    • Demonstrated significant differences in enzyme activity labeling between live and homogenized breast cancer cells.
    • Discovered several enzymes labeled in situ but not in vitro, suggesting context-dependent activity.

    Conclusions:

    • Optimized click chemistry provides a robust and sensitive method for in vivo activity-based protein profiling.
    • In situ enzyme activity profiling reveals distinct functional states compared to in vitro analysis.
    • This approach aids in identifying enzymes with unique activity profiles in living systems, relevant to disease states.