Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)--a general method for mapping sites of probe modification in proteomes.
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.
Weerapana
Eranthie
E
Department of Cell Biology, The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Speers
Anna E
AE
Cravatt
Benjamin F
BF
D000818:Animals; D001710:Biotin; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D015335:Molecular Probes; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods
D015335:Molecular Probes; D020543:Proteome; D001710:Biotin