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Nat Protoc. 2007;2(6):1414-25.

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

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Department of Cell Biology, The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.


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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