High-resolution functional proteomics by active-site peptide profiling

Abstract

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...

In-cell Selectivity Profiling of Serine Protease Inhibitors by Activity-based Proteomics

“…14-3-3 protein or glutathione S-transferase Omega 1; see supplemental Table T1. Therefore, we elected to restrict our analysis of potential targets (listed in Table I) to enzymes clearly belonging to the serine hydrolase class and to enzymes already identified by former activity-based proteomics studies using other FP probes on rodent (13,16,24,26) or human (7,27,45) cell lysates or tissue extracts. This validates the use of our novel FP-alkyne probe for the in situ detection of serine hydrolase activities in living cells.…”

Synthesis and Characterization of 5′-p-fluorosulfonylbenzoyl-2′ (or 3′)-(biotinyl)adenosine as an Activity-Based Probe for Protein Kinases

“…It is possible that by varying the size and nature of the linker, the reactivity and solubility of biotinyl-FSBA could be improved. [9][10][11] In general, we have found LC/MS techniques to be quite useful to directly compare the reactivity of the various forms of FSBA (unpublished data).…”

“…7 Several groups have described the use of activity-based probes (ABPs) to monitor the functional state of enzyme families in complex mixtures. The enzyme families for which ABPs have been developed include serine hydrolases, [9][10][11] cysteine proteases, 12,13 protein phosphatase, 14,15 metalloproteases, 16 and lipid kinases. 17 Earlier we described the use of 5´-flurosulfonylbenzoyl 5´-adenosine (FSBA) as a general ABP for the protein kinase family.…”

Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders. Proteomics approaches to the systems biology of cardiovascular diseases

“…These may not be distinguished by the standard methods described above. An approach referred to as ''activity-based protein profiling'' uses active site-directed probes to capture the active enzymes, but not the inactive or inhibited forms, for identification by MS or other means (36)(37)(38).…”