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Activity-Based Protein Profiling ANRV345-BI77-17 ARI 28 April 2008 15:9 ANNUAL REVIEWS Further Activity-Based Click here for quick links to Annual Reviews content online, including: Protein Profiling: From • Other articles in this volume • Top cited articles Enzyme Chemistry • Top downloaded articles • Our comprehensive search to Proteomic Chemistry Benjamin F. Cravatt,1 Aaron T. Wright,1 and John W. Kozarich2 1The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037; email: [email protected] 2Activx Biosciences, La Jolla, California 92037; email: [email protected] Annu. Rev. Biochem. 2008. 77:383–414 Key Words First published online as a Review in Advance on affinity label, mass spectrometry, proteomics March 26, 2008 Annu. Rev. Biochem. 2008.77:383-414. Downloaded from arjournals.annualreviews.org by Scripps Research Institute - KRESGE LIBRARY on 10/02/08. For personal use only. The Annual Review of Biochemistry is online at Abstract biochem.annualreviews.org Genome sequencing projects have provided researchers with a com- This article’s doi: plete inventory of the predicted proteins produced by eukaryotic 10.1146/annurev.biochem.75.101304.124125 and prokaryotic organisms. Assignment of functions to these pro- Copyright c 2008 by Annual Reviews. teins represents one of the principal challenges for the field of pro- All rights reserved teomics. Activity-based protein profiling (ABPP) has emerged as a 0066-4154/08/0707-0383$20.00 powerful chemical proteomic strategy to characterize enzyme func- tion directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes address- able by this method, and the biological discoveries attributable to its application. 383 ANRV345-BI77-17 ARI 28 April 2008 15:9 Genomic signatures, such as chromoso- Contents mal translocation and gene amplification, have provided some insights into protein ex- INTRODUCTION................. 384 pression (1, 2). Other genomic technolo- THE BASIC TECHNOLOGY gies, including transcriptional profiling and OF ABPP ........................ 386 RNA-interference-based gene silencing, have General Design Features provided insights into physiological and for ABPP Probes .............. 386 pathological processes, e.g., tumorigenesis (3, Analytical Platforms for ABPP .... 386 4), bacterial pathogenesis (5, 6), and insulin Types of Biological Experiments signaling (7). However, these methods rely that Can Be Performed on profiling and manipulation of gene ex- with ABPP .................... 390 pression to deduce protein function and do ENZYME CLASSES not generally capture the myriad of posttrans- ADDRESSABLE BY ABPP ...... 392 lational events that regulate protein activ- Serine Hydrolases ................ 392 ity (8). Sparked by this necessity, proteomics Cysteine Proteases ............... 396 has introduced several strategies for the Metallohydrolases ................ 398 global analysis of protein expression and func- Additional Classes of Hydrolases . 400 tion. Liquid chromatography-mass spectrom- Kinases and Nucleotide-Binding etry (LC-MS) platforms for shotgun analysis Proteins ...................... 400 (9, 10), yeast two-hybrid methods (11), and Glycosidases ..................... 405 protein microarrays (12) have greatly en- Cytochrome P450s ............... 406 riched our understanding of the expression Phosphatases ..................... 406 patterns, interaction maps, and in vitro func- Nondirected Probes that Target tional properties of proteins. However, an ac- Multiple Enzyme Classes ...... 407 curate assessment of the functional state of proteins in cells and tissues requires more direct methods to assess protein activity. Activity-based protein profiling (ABPP) has INTRODUCTION emerged as a key technology in the evolution Explosive advances in genomics have spawned of functional proteomics. multidisciplinary efforts to develop new tech- ABPP relies on the design of active-site- nologies for proteomics. To date, genome se- directed covalent probes to interrogate spe- quences of 639 organisms have been com- cific subsets (families) of enzymes in com- pleted, including three primates—rhesus, plex proteomes and to provide the basis for chimpanzee, and man. Over 2000 other a quantitative readout of the functional state genome projects are underway. This embar- of individual enzymes in the family. Proto- Annu. Rev. Biochem. 2008.77:383-414. Downloaded from arjournals.annualreviews.org by Scripps Research Institute - KRESGE LIBRARY on 10/02/08. For personal use only. rassment of riches for proteomics has put in typic ABPP probes target a large, but man- sharp relief the daunting challenge of assign- ageable, fraction of the enzyme proteome, ing functional, evolutionary, and interactive often defined by shared catalytic features. relationships to the full complement of pro- The size of the enzyme subset, however, can teins (the proteome) encoded by genomes. range from a few proteins to several hundred. The context and temporal variability of the ABPP probes utilize a range of chemical scaf- proteome distinguishes it from the back- folds, including mechanism-based inhibitors, ground of a relatively stable genome and lay- protein-reactive natural products, and general ers a manifold of complexity on the problem electrophiles. Because the activity state of an of developing large-scale methods for protein enzyme is regulated by a variety of posttrans- characterization. lational mechanisms (8, 13), chemical probes 384 Cravatt · Wright · Kozarich ANRV345-BI77-17 ARI 28 April 2008 15:9 that report on the structure and reactivity of wall biosynthesis. Using radiolabeled peni- enzyme active sites in cells and tissues can cillin G, whole cells or membrane fractions, provide high-content proteomic information and primitive gel electrophoretic methods by that is beyond the reach of standard expres- today’s standards, Strominger and colleagues sion profiling technologies. (23, 24) established that multiple penicillin- Although ABPP derives its enormous po- binding proteins (termed PBPs) are present tential from recent genomic advances, its in a bacterium. The number and function roots extend back for nearly a century. ABPP of these serine hydrolases vary in different evolved out of the preconvergence of organic bacteria. Using radiolabeled penicillin G as chemistry and enzymology (14). Motivated by a probe, they were subsequently able to pro- a desire to glean mechanistic and structural file the PBP selectivity of other penicillin and information on the nature of enzymatic catal- cephalosporin analogs and developed covalent ysis, enzyme chemists studied the modifica- affinity chromatography methods for captur- tion of purified proteins by reagents ranging ing and releasing PBPs for rapid purification from simple organic compounds to affinity la- (25, 26). Ultimately, this early approach linked bels to elaborate mechanism-based inhibitors. enzyme class function to biology by demon- With the goal of achieving a physical chemist’s strating that, by selectively inhibiting or mu- simplicity of experimental design, major ef- tating single PBPs, one could infer specific fort was focused on the up-front purification roles for PBPs in cell wall biosynthesis and of specific proteins by often heroic protocols cell shape determination (27). The limiting to isolate a few units of active enzyme for factor in these studies was the difficulty in ob- downstream modification and analysis. The taining protein sequence information and in results were impressive; the work of Balls & linking it to genomics information, which was Jensen (15–16) and their coworkers (17–19) nonexistent at that time. in the early 1950s on the stoichiometric inhi- The advances in protein separation tech- bition of the serine hydrolases, chymotrypsin, nology and mass spectrometry over the past trypsin, and cholinesterase by diisopropyl flu- 30 years, coupled with genomics, now allow orophosphate was relevant to the future de- routine downstream deconvolution of com- sign of ABPP probes. Subsequent work on plex proteomic samples. ABPP probes play a the irreversible inactivation of serine, cys- crucial role in fractionating these samples on teine, and threonine proteases by a wide vari- the basis of a key aspect of enzyme function— ety of molecules has been extensively reviewed catalytic activity. In addition, the interplay be- (20). tween new ABPP methods and the down- There is no doubt that early experiments in stream analytical technologies has created a the style of ABPP were performed before the synergistic cycle of advancement of both dis- word “proteomics” was invented. A case can ciplines. Todate, probes have been developed Annu. Rev. Biochem. 2008.77:383-414. Downloaded from arjournals.annualreviews.org by Scripps Research Institute - KRESGE LIBRARY on 10/02/08. For personal use only. arguably be made that the first such experi- for more than a dozen enzyme classes, in- ments were reported in the early 1970s using cluding proteases, kinases, phosphatases, gly- the classic serine-modifying antibiotic peni- cosidases, and oxidoreductases. They have cillin (21). Tipper & Strominger (22) had pro- contributed to our understanding of enzyme posed that the β-lactam nucleus of penicillin activity in specific physiological and patho- was a structural analog of the D-ala-D-ala ter- logical processes on a proteome-wide scale. minus of bacterial peptidoglycan and that the Uncharacterized enzymes for which no pre- chemically reactive β-lactam
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