Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes
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.
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In the first isoTOP-ABPP reports, an iodoacetamide probe carrying an alkyne handle was used to identify hyper-reactive cysteines across the proteome.
“In the first reports of the isoTOP-ABPP (isotopic tandem orthogonal proteolysis–activity-based protein profiling) approach, an iodoacetamide probe functionalized with an alkyne handle was used to identify hyper-reactive cysteines across the proteome176,177”
From Advances in covalent drug discovery (2022) · cites this paper as [176]
Recent activity-based protein profiling approaches use mass spectrometry and broadly reactive chemical probes to map allosteric sites in addition to active sites.
“Instead of focusing on active sites, more recent ABPP approaches use MS and broadly reactive chemical probes to also map allosteric sites176.”
From Advances in covalent drug discovery (2022) · cites this paper as [176]
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- Authors
- Weerapana, E., Speers, A. E., & Cravatt, B. F.
- Affiliations
- Scripps Research Institute (US)
- Corresponding author
- Cravatt, Benjamin F
- Venue
- Nature Protocols
- Published
- 2007-05-31
- Volume
- 2
- Issue
- 6
- Pages
- 1414-1425
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W1975857707
- Cited by
- 276
- References
- 28
- Citations by year
- 2026: 14 · 2025: 22 · 2024: 15 · 2023: 28 · 2022: 18 · 2021: 14 · 2020: 13 · 2019: 21 · 2018: 19 · 2017: 16 · 2016: 15 · 2015: 9 · 2014: 5 · 2013: 18 · 2012: 14
- FWCI
- 3.71
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute (R01CA087660)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- activity-based protein profiling, site mapping, chemical probes, enzyme activity, mass spectrometry, proteomics
- MeSH terms
- Animals, Biotin, Myocardium, Protein Conformation, Molecular Probes, Proteome, Gene Expression Profiling, Proteomics, Mice
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