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Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP) - a general method for mapping sites of probe modification in proteomes

Academic Article
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Overview

related to degree

  • Speers, Anna, Ph.D. in Chemistry, Scripps Research 2000 - 2005

authors

  • Weerapana, E.
  • Speers, Anna
  • Cravatt, Benjamin

publication date

  • 2007

journal

  • Nature Protocols  Journal

abstract

  • Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.

subject areas

  • Animals
  • Biotin
  • Gene Expression Profiling
  • Mice
  • Molecular Probes
  • Myocardium
  • Protein Conformation
  • Proteome
  • Proteomics
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Identity

International Standard Serial Number (ISSN)

  • 1754-2189

Digital Object Identifier (DOI)

  • 10.1038/nprot.2007.194

PubMed ID

  • 17545978
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Additional Document Info

start page

  • 1414

end page

  • 1425

volume

  • 2

issue

  • 6

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