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• W2116616991 abstract "Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3β-hydroxysteroid dehydrogenase/Δ5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(−)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments. Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3β-hydroxysteroid dehydrogenase/Δ5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(−)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments. Proteomic research aims to develop and apply methods to characterize the molecular and cellular function of the greater than 30,000 protein products encoded by the human genome (1.Anderson N.L. Anderson N.G. Proteome and proteomics: new technologies, new concepts, and new words.Electrophoresis. 1998; 19: 1853-1861Google Scholar, 2.Pandey A. Mann M. Proteomics to study genes and genomes.Nature. 2000; 405: 837-846Google Scholar). These efforts typically strive to provide a global analysis of either protein expression or protein function. Conventional approaches for the characterization of protein expression rely on two-dimensional gel electrophoresis, protein staining, and mass spectrometry (MS) 1The abbreviations used are: MS, mass spectrometry; 3HSD1, 3β-hydroxysteroid dehydrogenase/Δ5-isomerase-1; TriPS, trifunctional phenyl sulfonate probe; ER, estrogen receptor; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; NHS, N-hydroxysuccinimide; pPFK, platelet-type phosphofructokinase; PS-rhodamine, rhodamine-conjugated phenyl sulfonate probe; tTG, type II tissue transglutaminase; FTMS, Fourier transform MS; DHB, 2,5-dihydroxybenzoic acid. methods for the separation, detection, and identification of proteins, respectively (3.Corthals G.L. Wasinger V.C. Hochstrasser D.F. Sanchez J.C. The dynamic range of protein expression: a challenge for proteomic research.Electrophoresis. 2000; 21: 1104-1115Google Scholar). Although two-dimensional gel electrophoresis-MS methods are capable of determining the relative abundance and modification states of numerous proteins from endogenous sources (4.Nelson P.S. Han D. Rochon Y. Corthals G.L. Lin B. Monson A. Nguyen V. Franza B.R. Plymate S.R. Aebersold R. Hood L. Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.Electrophoresis. 2000; 21: 1823-1831Google Scholar), these approaches offer only an indirect estimate of protein function and may fail to detect critical posttranslational forms of regulation such as those mediated by protein-protein and/or protein-small molecule interactions (5.Kobe B. Kemp B.E. Active site-directed protein regulation.Nature. 1999; 402: 373-376Google Scholar). Recently strategies have emerged to profile the activity of enzyme superfamilies in complex proteomes using affinity-tagged chemical probes (6.Cravatt B. Sorensen E. Chemical strategies for the global analysis of protein function.Curr. Opin. Chem. Biol. 2000; 4: 663-668Google Scholar). These active site-directed probes profile proteins on the basis of function rather than abundance and are therefore capable of distinguishing, for example, active proteases from their inactive zymogens and/or inhibitor-bound forms (7.Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar, 8.Greenbaum D. Medzihradsky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 8: 569-581Google Scholar). To date, most efforts to create activity-based proteomic probes have exploited well known affinity labels as reactive groups, resulting in the generation of distinct sets of reagents that profile serine hydrolases (7.Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar, 9.Liu Y. Patricelli M.P. Cravatt B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14694-14699Google Scholar) and subclasses of cysteine proteases (8.Greenbaum D. Medzihradsky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 8: 569-581Google Scholar, 10.Faleiro L. Kobayashi R. Fearnhead H. Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.EMBO J. 1997; 16: 2271-2281Google Scholar). Recently serine hydrolase-directed probes were used to generate enzyme activity profiles that classified human breast and melanoma cancer cell lines into subtypes based on tissue of origin and state of invasiveness (11.Jessani N. Liu Y. Humphrey M. Cravatt B. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10335-10340Google Scholar), indicating that the information content achievable in activity-based proteomic experiments is of sufficient quantity and quality to depict higher order cellular properties. To accelerate the discovery of activity-based proteomic probes for enzyme classes lacking cognate affinity labeling reagents, we have introduced a non-directed or combinatorial strategy in which libraries of candidate probes are screened against complex proteomes for activity-dependent protein reactivity (12.Adam G.C. Cravatt B.F. Sorensen E.J. Profiling the specific reactivity of the proteome with non-directed activity-based probes.Chem. Biol. 2001; 8: 81-95Google Scholar, 13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar). Through a two-tiered strategy utilizing rhodamine-conjugated probes for rapid and sensitive target detection and biotin-conjugated probes for target isolation and molecular identification, members of a probe library bearing a sulfonate ester reactive group were found to label in an activity-based manner enzymes from at least six mechanistically distinct classes (13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar). During these studies, however, we noted that certain sulfonate targets evaded molecular characterization. These proteins tended to exhibit “difficult” properties, such as membrane association, context-dependent labeling, and/or co-migration with endogenous biotinylated proteins, that frustrated efforts to proceed from the stage of target detection to target identification. Accordingly we hypothesized that a method in which the target detection and target purification steps of activity-based proteomic experiments were consolidated might facilitate the characterization of such recalcitrant protein targets. Here we report the synthesis of a class of trifunctional chemical proteomic probes in which both rhodamine and biotin tags are coupled to a sulfonate ester reactive group, thereby permitting the simultaneous visualization and affinity isolation of activity-based protein targets by in-gel fluorescence scanning and avidin chromatography, respectively. Using these trifunctional probes, we report the molecular characterization of several protein targets previously resistant to characterization by the two-tiered strategy described above. These targets include the integral membrane enzyme 3β-hydroxysteroid dehydrogenase/Δ5-isomerase and two cofactor-dependent enzymes, platelet phosphofructokinase and type II tissue transglutaminase. Notably, the latter two enzymes were significantly up-regulated in the invasive estrogen receptor-negative (ER(−)) human breast cancer cell line MDA-MB-231 relative to non-invasive ER(+) cell lines MCF7 and T-47D. To permit the generation of libraries of trifunctional probes, a synthetic strategy was elaborated for the late stage incorporation of the reactive group. All reactions were carried out under an atmosphere of argon unless specified. Commercial reagents of high purity were purchased and used without further purification unless otherwise noted. To a solution of carboxylic acid (2) (Bachem, Torrance, CA; 0.06 g, 0.120 mmol, 1.0 equivalents (eq)) in N,N-dimethylformamide (3 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.032 g, 0.170 mmol, 1.4 eq) and N-hydroxysuccinimide (NHS) (0.032 g, 0.280 mmol, 2.3 eq). After stirring for 12 h at 25 °C, the reaction mixture was poured into saturated aqueous NaHCO3 solution (5 ml), and the product was extracted with ethyl acetate (3 × 5 ml). The organic layer was washed with water (15 ml) and saturated aqueous NaCl (15 ml), dried (MgSO4), and concentrated under reduced pressure. The crude NHS ester (0.070 g, 0.120 mmol, 3.5 eq) was dissolved in methanol (2 ml) followed by the addition of 5-(biotinamido)-pentylamine (Pierce; 0.015 g, 0.034 mmol, 1.0 eq). After stirring for 2 h at 25 °C, the solvent was evaporated under reduced pressure, and the remaining residue was washed with ethyl acetate (2 × 4 ml), solubilized in a minimal volume of chloroform, and transferred to a clean glass vial, and the solvent was evaporated. The process was repeated to rid the desired biotinylated intermediate of excess reagents and byproducts, affording 3 as a white film (50%): MALDI-FTMS (DHB) m/z 801.4007 (C41H58N6O7S + Na+ requires 801.3980). The 9-fluorenylmethoxycarbonyl protecting group was removed by the addition of morpholine (0.135 ml, 1.40 mmol, 100 eq) to a solution of 3 (0.012 g, 0.014 mmol, 1.0 eq) in N,N-dimethylformamide (1 ml). After stirring the reaction for 1 h at 25 °C, the solvent was removed under reduced pressure, and the product washed with ethyl acetate (2 × 3 ml). To a solution of the deprotected α-amino intermediate (0.010 g, 0.015 mmol, 1.1 eq) in N,N-dimethylformamide (1 ml) was added 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (Molecular Probes, Eugene, OR; 0.007 g, 0.014 mmol, 1.0 eq) and triethylamine (0.013 g, 0.130 mmol, 10.0 eq). After stirring at 25 °C for 2 h, the volatiles were removed by rotary evaporation, and high performance liquid chromatography (HPLC) purification afforded 4 (55%): MALDI-FTMS (DHB) 969.4870 (C51H68N8O9S + H+ requires 969.4902). The ε-amino tert-butoxycarbonyl protecting group was then removed by stirring 4 (0.006 g, 0.006 mmol, 1.0 eq) in 4 n HCl/dioxane (1 ml) for 1 h at 25 °C followed by evaporation of the volatiles under a stream of nitrogen. To a solution of the deprotected intermediate (0.005 g, 0.006 mmol, 1.0 eq) in methanol (1 ml) was added 5 (12.Adam G.C. Cravatt B.F. Sorensen E.J. Profiling the specific reactivity of the proteome with non-directed activity-based probes.Chem. Biol. 2001; 8: 81-95Google Scholar) (0.008 g, 0.017 mmol, 3.0 eq) and NaHCO3 (0.001 g, 0.012 mmol, 2.0 eq). After stirring for 4 h at 25 °C, the reaction was filtered, and the solvent was removed under reduced pressure. HPLC purification afforded the final product, the trifunctional phenyl sulfonate probe 1, or TriPS (40%): MALDI-FTMS (DHB) m/z 1179.5623 (C62H83N8O11S2+ requires 1179.5617). Mouse tissues were Dounce-homogenized in 50 mm Tris-HCl buffer, pH 8, 0.32 m sucrose, and the membrane and soluble fractions were separated by high speed centrifugation (sequential spins of 22,000 × g (30 min, pellet = membrane fraction) and 100,000 × g (60 min, supernatant = soluble fraction). The membrane fraction was washed twice and resuspended in Tris buffer without sucrose. Protein samples (2 mg/ml) were treated with 5 μm rhodamine-tagged or trifunctional sulfonate probe (250 μm stock in dimethyl sulfoxide), and the reactions were incubated for 1 h at 25 °C before quenching with 1 volume of standard 2× SDS-PAGE loading buffer (reducing). Quenched reactions were separated by SDS-PAGE (30 μg of protein/gel lane) and visualized in-gel using a Hitachi FMBio IIe flatbed laser-induced fluorescence scanner (MiraiBio, Alameda, CA). Labeled proteins were quantified by measuring integrated band intensities (normalized for volume). Breast cancer cell lines were grown to 80% confluency in RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum and harvested, sonicated, and Dounce homogenized in 50 mm Tris-HCl, pH 8.0 (Tris buffer). After centrifugation at 100,000 × g (40 min), the supernatant was collected as the soluble fraction, adjusted to 2 mg of protein/ml with Tris buffer, and labeled as described above. For affinity isolation of protein targets directly from tissue or cell line fractions, ∼8 mg of total protein was used as starting material (equivalent to ∼8 × 107 cells). Samples diluted to 2.5 ml with Tris buffer were labeled with the TriPS probe (5 μm) for 2.5 h at 25 °C and then applied to a PD-10 size exclusion column and eluted with 3.5 ml of Tris buffer. For unsolubilized membrane samples, Triton X-100 was added to a final concentration of 1.0%, and the samples were rotated for 1 h prior to passage over a PD-10 column and elution with Tris buffer with 0.1% Triton X-100. Desalted samples were fractionated by Q-Sepharose chromatography, and fractions containing the desired targets were affinity-isolated using avidin-agarose beads (Sigma) as described previously (7.Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar, 12.Adam G.C. Cravatt B.F. Sorensen E.J. Profiling the specific reactivity of the proteome with non-directed activity-based probes.Chem. Biol. 2001; 8: 81-95Google Scholar). Affinity-isolated proteins were separated by SDS-PAGE, excised from the gel, and digested with trypsin. The resulting peptides were analyzed by matrix-assisted laser desorption mass spectrometry (Kratos Axima CFR MALDI-TOF instrument, Kratos Analytical, Chestnut Ridge, NY). The MS data were used to search public data bases to identify the sulfonate-labeled proteins. cDNAs corresponding to each sulfonate target were purchased as expressed sequence tags (Invitrogen), sequenced, and transiently transfected into COS-7 cells following methods described previously (9.Liu Y. Patricelli M.P. Cravatt B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14694-14699Google Scholar). Transfected cells were harvested by trypsinization, resuspended in Tris buffer, sonicated, and Dounce-homogenized. The soluble fraction was separated by centrifugation at 100,000 × g (45 min), adjusted to 1 mg of protein/ml with Tris buffer, and labeled as described above. Soluble fractions of breast cancer cell lines (1 mg of protein/ml) were pretreated with 4 mm CaCl2 and/or GTP as indicated for 20 min at 25 °C followed by treatment with 250 μm 5-(and-6)-carboxytetramethylrhodamine-cadaverine (Molecular Probes). After incubation for 1 h at 25 °C, the reactions were quenched with 1 volume of standard 2× SDS-PAGE loading buffer (reducing) and separated by SDS-PAGE (15 μg of protein/gel lane). Samples were then visualized by in-gel fluorescence. For determination of the IC50 value for GTP inhibition of tTG activity, inhibition curves were generated for three distinct protein bands cross-linked to the rhodamine reporter group, and the estimated IC50 values from these curves were averaged to provide the reported value. To date, strategies for activity-based proteomics have typically utilized a two-tiered platform in which, first, proteomic samples are treated with fluorescently tagged chemical probes and separated by one-dimensional or two-dimensional gel electrophoresis, providing a rapid and sensitive method to detect labeled enzyme activities by in-gel fluorescence scanning (11.Jessani N. Liu Y. Humphrey M. Cravatt B. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10335-10340Google Scholar, 13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar, 14.Patricelli M.P. Giang D.K. Stamp L.M. Burbaum J.J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes.Proteomics. 2001; 1: 1067-1071Google Scholar, 15.Greenbaum D. Baruch A. Hayrapetian L. Darula Z. Burlingame A. Medzhiradsky K. Bogyo M. Chemical approaches for functionally probing the proteome.Mol. Cell. Proteomics. 2002; 1: 60-68Google Scholar). A second series of proteome labeling experiments using biotinylated probes is then required to permit the affinity isolation of protein activities by avidin chromatography procedures. The molecular identity of the purified protein activities can then be determined by standard tryptic digestion-mass spectrometry techniques. For low abundance targets of chemical proteomic probes, an anion exchange chromatography step may be required to enrich these proteins prior to avidin-based affinity purification. In the course of screening cell and tissue proteomes with rhodamine-tagged sulfonate probes, we detected several labeled protein activities for which molecular identities were sought. Although many of these sulfonate-reactive proteins could be affinity purified with biotinylated probes, permitting their identification by mass spectrometry methods (13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar), some sulfonate targets proved resistant to molecular characterization by these methods. These sulfonate-reactive proteins generally represented lower abundance targets that displayed one or more of the additional challenging properties: 1) membrane association, 2) context-dependent labeling, and/or 3) migration on SDS-PAGE in the vicinity of endogenous biotinylated proteins. For example, an analysis of a panel of human breast carcinoma cell lines uncovered two 75–80-kDa phenyl sulfonate-reactive proteins enriched in the ER(−) invasive line MDA-MB-231: an 80-kDa protein that exhibited ATP-sensitive labeling (Fig. 1, single arrowhead) and a 75-kDa protein that displayed calcium-dependent labeling (Fig. 1, double arrowhead). Initial attempts to label and affinity-isolate these proteins with biotinylated probes, either directly from the crude cytosolic preparation or following prefractionation by Q anion exchange chromatography, were unsuccessful. One challenge facing these analyses was that the 75–80-kDa sulfonate targets migrated in the vicinity of endogenous biotinylated proteins (7.Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar, 14.Patricelli M.P. Giang D.K. Stamp L.M. Burbaum J.J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes.Proteomics. 2001; 1: 1067-1071Google Scholar), complicating target detection by avidin blotting methods. Additionally we noted that both the 75- and 80-kDa proteins were unreactive with the phenyl sulfonate probe following desalting of the crude proteomic preparation (Fig. 2A), indicating that these proteins required additional cytosolic factors to maintain activity. This context-dependent reactivity displayed by the 75–80-kDa proteins precluded their enrichment by chromatography methods prior to probe labeling. Attempts to label cytosolic preparations with biotinylated sulfonate probes prior to Q chromatography and then analyze the resulting column fractions by avidin blotting were hindered by the limited sensitivity, dynamic range, and throughput of this screening method (data not shown). To circumvent these shortcomings, a trifunctional probe was synthesized in which both rhodamine and biotin substituents were coupled to the phenyl sulfonate ester reactive group (TriPS, 1, see Scheme 1). We anticipated that this TriPS probe would allow us to track probe-labeled proteins through fractionation and purification protocols using in-gel fluorescence scanning, a method that offers greater sensitivity, dynamic range, and throughput relative to avidin blotting methods (13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar, 14.Patricelli M.P. Giang D.K. Stamp L.M. Burbaum J.J. Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes.Proteomics. 2001; 1: 1067-1071Google Scholar, 15.Greenbaum D. Baruch A. Hayrapetian L. Darula Z. Burlingame A. Medzhiradsky K. Bogyo M. Chemical approaches for functionally probing the proteome.Mol. Cell. Proteomics. 2002; 1: 60-68Google Scholar). Additionally, fluorescence detection was expected to assist in identifying sulfonate targets in regions of the SDS-PAGE-fractionated proteome, like the 75–80-kDa range, that are complicated by the presence of endogenous biotinylated proteins.Fig. 2Labeling and affinity enrichment of protein targets with trifunctional chemical proteomic probes.A, heat-sensitive sulfonate targets in the MDA-MB-231 soluble proteome are labeled by both the rhodamine-tagged (PS-Rhodamine) and trifunctional (TriPS) phenyl sulfonate probes. Desalting the proteome prior to treatment with probe blocks the labeling of both the calcium-dependent 75-kDa and ATP-sensitive 80-kDa sulfonate targets. Δ, heat-denatured proteome. B, pretreatment of the MDA-MB-231 soluble proteomic fraction with TriPS followed by Q-Sepharose anion exchange chromatography and SDS-PAGE analysis identifies fractions enriched for the 75- and 80-kDa sulfonate targets (F 9–15). Avidin-based affinity purification greatly enriched these TriPS-labeled targets from the pooled Q fractions (right panel). Fluorescent gel images are shown in grayscale.View Large Image Figure ViewerDownload (PPT)Scheme 1Synthesis of TriPS probe.FMOC, 9-fluorenylmethoxycarbonyl; BOC, tert-butoxycarbonyl; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMF, N,N-dimethylformamide; NHS-TAMRA, N-hydroxysuccinimidyl ester of tetramethylrhodamine; Biotin-NH2, 5-biotinamidopentylamine; Et3N, triethylamine.View Large Image Figure ViewerDownload (PPT) The trifunctional probe TriPS was synthesized as described under “Experimental Procedures” and in Scheme 1. Briefly, lysine differentially protected on the ε- and α-amino groups (2) was selected as the starting material due to the presence of three sites for attachment through the formation of amide bonds. Compound 3 was formed through coupling of 5-biotinamidopentylamine with the activated ester of 2. Deprotection of the α-amino group and reaction with the N-hydroxysuccinimidyl ester of tetramethylrhodamine resulted in formation of the protected bifunctional linker 4. Removal of the tert-butoxycarbonyl protecting group followed by reaction with the preformed NHS ester of 10-(benzenesulfonyl)oxodecanoic acid (5) (12.Adam G.C. Cravatt B.F. Sorensen E.J. Profiling the specific reactivity of the proteome with non-directed activity-based probes.Chem. Biol. 2001; 8: 81-95Google Scholar) and subsequent HPLC purification provided the TriPS probe (1). From compound 4, additional trifunctional sulfonate probes were also synthesized (data not shown). The TriPS probe showed an overall proteome reactivity profile similar to the original rhodamine-tagged phenyl sulfonate probe (PS-rhodamine), labeling both the 75- and 80-kDa targets of interest (Fig. 2A). The labeling intensity of these proteins by the TriPS probe was moderately reduced compared with the reactivity observed with the parent PS-rhodamine probe, possibly due to the increased steric bulk of the trifunctional agent. Treatment of MDA-MB-231 cytosol (2.5 ml at 3.5 mg/ml) with the TriPS probe, followed by Q chromatography and SDS-PAGE analysis of the resulting fractions, provided a straightforward method by which to visualize fractions that were enriched for the labeled 75- and 80-kDa targets (Fig. 2B). These fractions were combined and treated with avidin-agarose beads as described previously (7.Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar, 12.Adam G.C. Cravatt B.F. Sorensen E.J. Profiling the specific reactivity of the proteome with non-directed activity-based probes.Chem. Biol. 2001; 8: 81-95Google Scholar). Elution of bound proteins by heating in 1 volume of standard SDS-PAGE loading buffer provided a greatly enriched sample of TriPS-labeled targets (Fig. 2B). Protein bands corresponding to the 75- and 80-kDa targets were excised from the gel, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry, resulting in their identification as tTG and platelet-type phosphofructokinase (pPFK), respectively. A more detailed characterization of these sulfonate targets is described below. Collectively these results highlight the value of trifunctional chemical probes as tools that simplify the transition from target detection to target identification in activity-based proteomic experiments. To test whether the devised methods would apply to the isolation and characterization of membrane-associated as well as soluble proteins, we pursued the identification of a 40-kDa phenyl sulfonate target selectively expressed in mouse testis membranes (13.Adam G. Sorensen E. Cravatt B. Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.Nat. Biotechnol. 2002; 20: 805-809Google Scholar). Following treatment with the TriPS probe, testis membrane proteins were solubilized with Triton X-100 and separated by Q chromatography (Fig. 3A). Fractions enriched in the 40-kDa sulfonate target were subjected to avidin-based affinity purification procedures, and the enriched protein was identified by MALDI peptide mapping as 3β-hydroxysteroid dehydrogenase/Δ5-isomerase-1 (3HSD1), an NAD+-dependent integral membrane protein found predominantly in the gonads and adrenal gland (16.Bain P. Yoo M. Clarke T. Hammond S. Payne A. Multiple forms of mouse 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isom" @default.
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• W2116616991 title "Trifunctional Chemical Probes for the Consolidated Detection and Identification of Enzyme Activities from Complex Proteomes" @default.
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