Abstract: The present invention provides compositions and methods for assessing profiles of catalytically active enzymes in compositions containing a plurality of proteins. In preferred embodiments, the enzyme is a hydrolase, most preferably a cysteine protease. The methods described herein use activity based probes (“ABPs”) that have an affinity moiety for directing the binding of the ABP to one or more catalytically active target enzymes, a reactive group for forming a covalent bond at an active site of the target enzyme(s), and a TAG (e.g., a detectable label, preferably a fluorophore). One or more ABPs may be combined with a protein-containing sample under conditions for binding and reaction of the ABP(s) with target enzyme(s) that are present in the sample. The resulting products may then be used to assess the active enzyme profile of the sample, and can be correlated to the presence, amount, or activity of one or more target enzyme(s) present in the original complex protein mixture.

Abstract: The present invention provides tagged acyl phosphate probes (“TAPPs”), and methods of their preparation and use. The subject methods and compositions can provide enhanced simplicity and accuracy in identifying changes in the presence, amount, or activity of target proteins in a complex protein mixture, preferably nucleotide binding proteins using nucleotide binding protein-directed TAPPs. The profiling methods described herein can have a number of steps leading to the identification of target nucleotide binding protein(s) in a complex protein mixture.

Abstract: Methods and compositions are described for analyzing complex protein mixtures, such as proteomes, using activity-based probes. In particular, probes that specifically react with and bind to the active form of one or more target proteins are employed. Labeled peptides obtained from the labeled active target proteins can be used in screening and identification procedures, and can be related to the identity, presence, amount, or activity of active members of the desired target protein class. The methods and compositions described herein can be used, for example, to provide diagnostic information concerning pathogenic states, in identifying proteins that may act as therapeutic targets, and in drug discovery.

Abstract: Methods and compositions are described for analyzing complex protein mixtures using fluorescent activity-based probes. In particular, probes that specifically react with and bind to the active form of one or more target proteins are employed. Fluorescent signals obtained from the labeled active target proteins can be related to the presence or amount of active members of the desired target protein class. The methods and compositions described herein can be used, for example, to provide diagnostic information concerning pathogenic states, in identifying proteins that may act as therapeutic targets, and in drug discovery.

Abstract: The invention relates to compositions and methods for the synthesis and use of Adenosine nucleotide binding protein-directed affinity labels. Adenosine nucleotide binding proteins may be labeled with probes comprising adenosine, or an analogue thereof, functionalized at the 5′ position with reactive group capable of reacting with an amino acid side chain functionality at an adenosine nucleotide binding site, and at the 2′ or 3′ position with a TAG for sequestering and/or identifying the resulting conjugate. The probes may be used for determining the presence or amount of one or more adenosine nucleotide binding proteins in a complex mixture, particularly a cellular mixture, for screening for drugs, and other purposes associated with the presence of the adenine nucleotide-binding protein(s) in a cell or changes in the presence, amount, activity, or relative concentration of the active adenosine nucleotide-binding protein.