Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.capillary electrophoresis ͉ fluorescence ͉ MS ͉ protease T he completion of several major genome sequencing projects has both accelerated the pace and broadened the scale of modern biology. For example, it is estimated that a complete molecular understanding of the human organism will require the characterization of at least 100,000 proteins (1). Several innovations in the field of proteomics have advanced to meet the demands of postgenome biology. Particularly, improvements in the resolving capacity and reproducibility of 2D gel electrophoresis (2) and the increased sensitivity and accuracy of modern MS methods (3) have enabled the global characterization of protein expression levels in complex biological systems. Nonetheless, the large dynamic range of protein expression in biological tissues and fluids renders these methods ineffective at detecting and͞or quantitating low-abundance proteins in the absence of labor-intensive prefractionation or enrichment procedures (2, 4). Moreover, abundance-based proteomic strategies fail to provide direct information regarding protein function or activity.To address these limitations, a chemical proteomic technology called activity-based protein profiling (ABPP) (5) has emerged that utilizes active site-directed probes to enable the parallel measurement of the activity of many enzymes in complex proteomic mixtures (5-8). Activity-based probes (ABPs) enable the detection of changes in enzyme activity independent of alterations in protein abundance, and provide a specific enrichment strategy for low-copy-number enzymes without interference from more abundant proteins. To date, ABPP has relied on gel-based methods for proteome analysis that are difficult to automate and often fail to resolve highly related protein species (5, 6, 9, 10). Here, we describe a gel-free platform for ABPP, termed Xsite, that permits the rapid and systematic quantification and identification of enzyme activities in complex protein mixtures. Materials and MethodsGeneral Materials and Methods. Fluorophosphonate probes were synthesized as described (5). Synthesis of the cathepsin probe, AX6429, is detailed in supplemental materials (Fig. 3, which is published as supporting information on the PNAS web site). Rat FAAH was expressed in Escherichia coli and purified as described (11). Porcine trypsin and equine butyrylcholinesterase were p...

all-trans-Retinoic acid is known to bind to the retinoic acid receptors (RARs) resulting in an increase in their transcriptional activity. In contrast, recently identified 9-cis-retinoic acid (9-cis-RA), which is an additional endogenous RA isomer, is capable of binding to both RARs and retinoid X receptors (RXRs). These distinct properties have raised questions as to the biological role governed by these two retinoic acid isomers and the set of target genes that they regulate. Herein, we report the synthesis of high specific activity [3H]-9-cis-RA and its application to study the ligand-binding properties of the various retinoid receptor subtypes. We examined the binding properties of RARs and RXRs for a series of synthetic retinoids and compared the ligand-binding properties of these arotinoid analogs with their ability to regulate gene expression via the retinoid receptors in a cotransfection assay. The utilization of the [3H]-9-cis-RA competitive binding assay and the cotransfection assay has made it possible to rapidly identify important structural features of retinoids leading to increased selectivity for either the RAR or RXR receptor subtypes.

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

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