An Immuno-Chemo-Proteomics Method for Drug Target Deconvolution Chaitanya Saxena,* Eugene Zhen, Richard E. Higgs, and John E. Hale* Integrative Biology, Greenfield Laboratories, Eli Lilly and Company, Greenfield, Indiana 46140 Received March 24, 2008
Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-R inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-R, GSK3-β, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-R, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix. Keywords: chemical proteomics • Bisindolylmaleimide III • PKC inhibitor • drug target deconvolution • peptide coupled small molecule • mass spectrometry • affinity chromatography
Introduction In the postgenomic era, the perceived ‘failure’ of target-based drug discovery1 has led to a resurgence of system-biology based tools2 where test compounds are screened based on their ability to elicit phenotypic changes in model cell and evolutionary lower organism systems. This approach of ‘phenotypic screening’ poses new challenges of identifying targets which are responsible for observed phenotypic changes upon test compound administration. The process of identifying targets, from possibly several thousands of biomolecules working in the cell, is termed as target deconvolution.2 Identification of targets is important for elucidating the biological mechanism of disease, mechanism of drug action, rational drug design, and patient stratification. In addition, it also provides a target-specific toxicity profile for given test compounds. Chemical proteomics is one of the many techniques for target deconvolution where affinity chromatography is used to identify the cellular target proteins that interact with small molecules.3 Traditionally, a small molecule of interest is modified in such a way that it can be immobilized on a solid support through a hydrophilic linker. After incubating this * To whom correspondence should be addressed. Chaitanya Saxena: phone, 317-651-1539; fax, 317-277-0173; e-mail,
[email protected]. John E. Hale: phone, 317-277-5373; fax, 317-277-0173, e-mail, hale_john_e@ lilly.com.
3490 Journal of Proteome Research 2008, 7, 3490–3497 Published on Web 07/01/2008
immobilized small molecule with protein extract for a brief period, a series of washing steps are performed to remove the unbound protein from the mixture. At this stage, specific buffer conditions capable of disrupting small molecule-protein interactions are applied to recover the target proteins. At the end, proteins are typically identified by mass spectrometry or immunodetection methods. Although this approach provides some level of target deconvolution and has been successfully used,3,5 it remains limited because of the nature of the probe where ‘solid support’ is used to immobilize the small molecules. Problems associated with steric hindrance6 and limited mobility of the probe molecule may alter its binding to targets. A large resin surface area also increases the chance of nonspecific binding and may lead to the identification of false positives. Coupling small molecules with solid supports makes it difficult to characterize binding affinity toward the protein targets after immobilization. Typically, immobilized molecules on solid support exhibit weaker affinity for target proteins compared to their free state. This could lead to unacceptable losses of target proteins during the washing steps. To circumvent some of these shortcomings associated with the small molecule/solid support approach, we developed a generalized strategy for target deconvolution. Here, we describe the development and application of an orthogonal technique for target deconvolution where the test molecule Bisindolyl10.1021/pr800222q CCC: $40.75
2008 American Chemical Society
Immuno-Chemo-Proteomics For Target Deconvolution maleimide III (Bis-III), a derivative of a known Protein Kinase C (PKC) inhibitor,7 was coupled with FLAG peptide. The FLAGtagged kinase inhibitor was demonstrated to retain inhibitory activity and was then incubated with cell lysate. Inhibitor-protein complexes were isolated with an anti-FLAG affinity resin. In comparison, the same compound was immobilized to a solid support using the traditional approach, and was then incubated with cell lysate. We provide a comparative analysis of both techniques. The development of the peptide-coupled-smallmolecule approach provided us an extra peptide-handle to perform various related but essential tasks of binding affinity measurements and target interaction validation studies. In the process, we not only could identify previously unknown targets of Bis-III but also established a robust process for deconvoluting targets from seemingly uncountable false positives originating from different types of probes.
Material and Methods Reagents. Cell culture media was purchased from Invitrogen (Carlsbad, CA). Epoxy-activated Sepharose 6B resin was obtained from Amersham Biosciences (GE Healthcare) and BisIII was purchased from Alexis Biochemical (San Diego, CA). Modified FLAG peptide (NH2-DYKDDDDKC-COOH) with an extra cysteine residue at the C-terminal end was customsynthesized at Genscript, Inc. (NJ). A heterobifunctional crosslinker succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate] (LC-SMCC) capable of linking a sulfhydryl group at one end and an amine at the other end was purchased from Pierce (Rockford, IL). Protease inhibitor cocktail was purchased from Calbiochem (EMD Biosciences, San Diego, CA). All other reagents were obtained from Sigma (St. Louis, MO). Antibodies used were rabbit polyclonal anti-PKC-R antibody (Cell Signaling Technology, Beverly, MA), mouse monoclonal anti-Glycogen Synthase Kinase 3 R/β (GSK3 R/β) antibody (Abcam, Cambridge, MA). Agarose based anti-FLAG M2 Affinity Gel was purchased from Sigma. Active recombinant Glycogen Synthase Kinase 3β (GSK-3 β) was purchased from Biovision Research Product (Mountain View, CA). Tau protein, as GSK-3 β substrate and Phospho-Tau specific antibody were purchased from Sigma. Anti-FLAG M2 affinity resins were purchased from Sigma. Preparation of FLAG-Coupled-Bis-III. Stock solutions of 13 mM of Bis-III and LC-SMCC were prepared in DMSO. Final concentrations of 1 mM Bis-III and 1 mM LC-SMCC were added to 0.6 M phosphate buffer (pH 7.2) in a 1 mL preparation. The reactant solution was kept under constant orbital mixing for 45 min in the dark at room temperature. From stock solution of 13 mM, final concentration of 1 mM of modified FLAG peptide (NH2-DYKDDDDKC-COOH) was added to the solution and mixing was continued for another 45 min. The resultant solution was subjected to gel-filtration using Sephadex G-25 (Sigma) to remove unreacted Bis-III and LC-SMCC. Gelfiltration flow-through containing unreacted FLAG, FLAG reacted to LC-SMCC and FLAG coupled with Bis-III through LC-SMCC were separated over a 250 × 4.6 mm Kromasil C18 HPLC column using a linear gradient of water/acetonitrile from 95:5 to 55:65 developed over a period of 45 min with a flow rate of 1 mL/min using an Agilent 1100 HPLC pump. Fractions were collected every 3 min and were analyzed using a MALDITOF (4700 Proteomic analyzer, Applied Biosystems). Fractions showing peaks at m/z 1832.83 were pooled together and lyophilized. The concentration of thus obtained ‘FLAG-coupled-
research articles Bis-III’ was measured by acquiring absorption spectra of the Bis-III, which is the only optically active moiety in the visible spectral range. Immobilization of Bisindolylmaleimide-III on Solid Support. Immobilization of Bis-III on a solid support was accomplished as described.8 Briefly, epoxy-activated 6B resins were washed extensively using distilled water and were resuspended in 2 vol of 20 mM Bis-III dissolved in 50% dimethylformamide/0.1 M Na2CO3, pH 11. NaOH was added to the reaction mixture to 10 mM final concentration and coupling was performed overnight in the dark at room temperature with constant orbital mixing. The next day, resins were washed five times with 50% dimethylformamide/0.1 M Na2CO3 to remove unbound Bis-III. Remaining epoxy reactive groups on resins were blocked by adding 1 M ethanolamine, pH 11, and continuing the orbital shaking for another 4 h. Resins were finally washed with at least three cycles of alternating pH as per the manufacturer’s instruction. Each cycle consisted of a wash with 0.1 M acetate buffer, pH 4.0, containing 0.5 M NaCl followed by a wash with 0.1 M Tris-HCl buffer, pH 8.0, containing 0.5 M NaCl. Control-resins were prepared by directly incubating the drained epoxy-activated Sepharose 6B resins with 1 M ethanolamine and were washed as described above. The beads were used right after preparation. Cell Culture and Lysis. HeLa cells were cultured in a bioreactor to yield more than 1 kg of wet weight cells for various purposes. Cells were frozen immediately after harvesting and were thawed a day before the lysis at 4 °C in an ice-water mixture. Ten grams of wet weight cells was lysed using a Dounce homogenizer in buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.25% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM DTT, and protease inhibitor cocktail. Lysate was centrifuged at 500g for 15 min at 4 °C to remove the cellular debris. Pellets were discarded and the supernatant was further centrifuged at 40 000g at 4 °C. Pellets were discarded and supernatant thus obtained was aliquoted, quickly frozen in liquid nitrogen and stored at -70 °C. Protein concentration of the aliquot was measured using the Bradford method.9 On the day of the experiment, required numbers of aliquots were thawed in ice at 4 °C. To get final protein concentration of 2 mg/mL, 128 µL of protein stock aliquots (15.6 mg/mL) was diluted to 1000 µL in high salt buffer (50 mM Hepes (pH 7.5), 1 M NaCl, 0.1% NP-40, 1 mM EDTA) immediately before the in vitro association experiments. Two milligrams of protein was used for all subsequent experiments. Affinity Chromatography with FLAG-Coupled-Bis-III. A hundred micromolar of FLAG-coupled-Bis-III or control-FLAG peptide (NH2-DYKDDDDKC-COOH) was incubated with 1 mL of high salt cell lysate for 2 h at 4 °C. In the preincubation experiments, 1 mM free Bis-III was added to the lysates prior to the addition of FLAG-coupled-Bis-III. After incubation, 200 µL of drained anti-FLAG M2 affinity resin washed with the high salt buffer was added to the mixture. The mixtures were incubated overnight at 4 °C with constant orbital mixing. The next day after brief centrifugation, the supernatant was collected and the anti-FLAG affinity resins were washed four times using 500 µL of the high salt buffer each time. The resins were eluted with 3 vol of elution buffer containing either (a) 1 mM Bis-III in high salt buffer, (b) 10 mM FLAG peptide in high salt buffer, or (c) 0.1 M Glycine (pH 3.5). Because of poor solubility of Bis-III in aqueous solution, a stock solution of Bis-III was prepared in DMSO and the required amount of Bis-III was Journal of Proteome Research • Vol. 7, No. 8, 2008 3491
research articles diluted in the high salt buffer to prepare the elution buffers; thus, elution buffer contained less than 1% of DMSO. Affinity Chromatography with Resin Immobilized Bis-III. A hundred microliters of Bis-III-resins or control-resins with no Bis-III immobilized was equilibrated with high salt buffer and later incubated with 1 mL of cell lysate in high salt buffer for 3 h at 4 °C. In preincubation experiments, 1 mM free BisIII was added to the lysates prior to the addition of Bis-III resins. Resins were washed four times using 500 µL of the high salt buffer each time. The resins were eluted with 3 vol of elution buffer containing 1 mM Bis-III in 50 mM Hepes (pH 7.5), 1 M NaCl, 0.1% NP-40, and 1 mM EDTA. Protein Sample Preparation for Analysis. Eluted proteins were concentrated in a Speed-Vac system and were subsequently precipitated using a chloroform-methanol precipitation method.10 Precipitated proteins were dissolved in the LDS sample buffer (Invitrogen), separated using 4-12% SDS/PAGE gel, and visualized using Coomassie blue stain or silver stain or were transferred to a nitrocellulose membrane and immunoblotted with the specific antibodies. Mass Spectrometry. Coomassie-stained gel bands from the whole lanes were cut into 1 mm slices and the proteins were reduced, alkylated and trypsin-digested.11 Peptides were extracted from the gel by incubating the trypsin-digested bands with 1 M urea in 50 mM NH4HCO3. Peptides were desalted and concentrated using µ-C18 Ziptips (Millipore). Peptide fragments thus obtained were injected onto a 5 cm × 7.5 µm C-18 reverse-phase column, and were eluted with a gradient of 5-50% CH3CN developed over 60 min. the eluate was infused into an IT mass spectrometer (LTQ, Thermo Finnigan) using a nanoelectrospray source. and data were collected in the triple-play mode. MS/MS spectra were searched against a nonredundant protein database with SEQUEST and X! Tandem for the identification of proteins.12 Supplementary Information pages provide the details of the protein identification process. In Vitro Kinase Assay. GSK3-β inhibition assay was performed at 37 °C in a total volume of 20 µL. One microgram of active recombinant GSK3-β protein was taken up in 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerol phosphate, 12 mM MgCl2, 2 mM DTT, 0.1 M Na3VO4, and 200 µm ATP and this mixture was incubated with varying concentration of Bis-III and FLAGcoupled-Bis-III for 5 min. Tau protein (100 ng) was used as the substrate for GSK3-β. The kinase reaction proceeded for 30 min at 37 °C. Afterward, LDS running buffer was added to the mixture and proteins were separated over SDS-PAGE. Proteins were transferred to a PVDF membrane and immunoblotted with anti-phosphoTau antibody. The primary antibodies was rabbit anti-human-phospho-Tau (pSer396 and pSer202) (Sigma, 1:100) and secondary antibody was FITC-conjugated anti-rabbit IgG (Sigma, 1:100). Membrane was imaged for the specific fluorophore using a Li-Cor Odyssey IR imaging system. Decrease in fluorescence intensity with increasing concentration of Bis-III and/or FLAG-coupled-Bis-III represented inhibition of GSK3-β kinase activity and was quantified to obtain the IC50 values.
Results Development and Characterization of a Soluble Peptide Coupled Small Molecule Probe. As an alternative to immobilizing Bis-III on solid support, we sought an affinity handle which could carry the small molecule in a freely diffusable form. The other condition in handle selection was that it should be easily isolatable once it has interacted with the protein matrix. 3492
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Figure 1. Preparation and characterization of FLAG-coupled-BisIII probe. (A) Structure of PKC-R inhibitor Bis-III and the linker (LC-SMCC) used to couple Bis-III with the modified FLAG peptide. (B) Mass spectrum of the purified FLAG-coupled-Bis-III showing m/z peak at 1832.83. (C) GSK3-β kinase activity inhibition assay in presence of Bis-III and FLAG-coupled-Bis-III.
FLAG-tag, or FLAG octapeptide (NH2-DYKDDDDK-COOH), is a polypeptide protein tag that is often added to a recombinant expressed protein for identification and purification purposes.13,15 The epitope of FLAG-tag and commercially available anti-FLAG antibody are well-characterized and researchers use it for a wide variety of purposes. Thus, we selected FLAG-tag as the “handle” to be attached to Bis-III. A modified form of the FLAGtag where a cysteine residue was added to its C-terminus was custom-synthesized. A heterobifunctional linker LC-SMCC was used to couple the primary amine group of Bis-III with the sulfhydryl of the modified FLAG-peptide. Increased hydrophilicity to reduce nonspecific binding and extended spacing arm of 16.2 Å for efficient capture of target proteins dictated the choice of linker in the form of LC-SMCC. Figure 1A show the coupling scheme of the modified FLAG-tag with Bis-III. To reduce the reaction of the primary amine of the modified FLAGpeptide with the NHS moiety of the LC-SMCC, Bis-III was reacted first with LC-SMCC and the modified FLAG-peptide was added later in the reaction (see Material and Methods). FLAG-coupled-Bis-III was purified by gel-filtration chromatography followed by RP-HPLC. Coupling was characterized by acquiring the mass spectra of the purified FLAG-coupled-BisIII which displays a m/z value at 1832.83 (Figure 1B). To ascertain that FLAG-coupled-Bis-III was still an active kinase inhibitor, a kinase activity inhibition assay of GSK3-β was
Immuno-Chemo-Proteomics For Target Deconvolution
Figure 2. Elution profile of proteins from anti-FLAG antibody resins. Different elution conditions were applied for the elution, and the eluted proteins were concentrated before SDS-PAGE analysis. Flow through is the unbound fraction to FLAG-coupledBis-III. Proteins were separated over SDS-PAGE and visualized using Coomassie blue stain. Corresponding samples were analyzed with immunoblotting technique using a monoclonal antibody against GSK3-R/β protein.
performed in the presence of free Bis-III or FLAG-coupled-BisIII since GSK3-β is a known target of Bis-III.16,17 Free Bis-III inhibited GSK3-β with an IC50 value of ∼0.14 µm. The FLAGcoupled-Bis-III inhibited GSK3-β with an IC50 value of 4 µm (Figure 1C). Although FLAG-coupled-Bis-III showed lower kinase inhibition efficiency, we concluded that the interaction of FLAG-coupled-Bis-III with its binding partner would be retained when we incubated this probe with the protein matrix. Therefore, we knew the exact probe used to isolate protein binding partners was active. Optimization of Affinity Chromatography and Capture of Cellular Binding Partner Using FLAG-Coupled-Bis-II. The assumption that, with 4 µm IC50 value, FLAG-coupled-Bis-III will still bind to its target proteins was confirmed by performing in vitro association experiments as shown in Figure 2. GSK3-β in the HeLa total cell lysate specifically interacted with the probe and was enriched after anti-FLAG chromatography, while no binding was visible in the control experiments where BisIII was not conjugated to FLAG peptide. Preincubation of cell lysate with 1 mM Bis-III prior to the addition of FLAG-coupledBis-III eliminated the binding of GSK3-β to the FLAG-coupledBis-III probe (data not shown). In optimizing affinity chromatographic conditions, we found that the amount of antibody and the incubation time were critical in capturing all FLAGcoupled-Bis-III probes once it had interacted with the protein matrix. Optical absorption of Bisindolylmaleimide was measured in the supernatant to conclude that FLAG-coupled-BisIII has been completely captured by anti-FLAG antibody (data not shown). Once the FLAG-coupled-Bis-III along with the interacting proteins was bound to anti- FLAG resins, there were multiple options for elution. We eluted bound proteins using
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Figure 3. Comparative elution profile of proteins using two techniques. Proteins were eluted after affinity chromatography and concentrated 100-fold to cell lysate and flow through. Proteins were separated over SDS-PAGE and visualized using silver stain. Corresponding samples were analyzed with immunoblotting technique using monoclonal antibody against GSK3R/β and PKC-R.
only 1 mM free Bis-III, or eluated the whole FLAG-coupledBis-III-protein complex with 10 mM modified-FLAG peptide or 100 mM glycine (pH 3.5). All eluted proteins were separated over SDS-PAGE and visualized using Coomassie blue stain (Figure 2). Although the starting protein concentration, the amount of FLAG-coupled-Bis-III probe, the amount of FLAG antibody resins and buffer conditions were the same, the eluted protein profiles using different elution buffer was different (Figure 2). A 1 mM Bis-III brought most proteins off the antiFLAG resins; however, the amount of protein eluted from the control in a similar experiment was also higher. Overall, we found that all of these conditions recovered the known target GSK3-β from the anti-FLAG resins, as shown by immunoblotting in Figure 2. After establishing that FLAG-coupled-Bis-III probe can capture the target protein and 1 mM Bis-III in elution buffer can elute most of the proteins off the anti-FLAG resin, we repeated this experiment three times. Eluted proteins from each replicate sample were separated over SDS-PAGE and identified using either Western blot or mass spectrometric techniques. Whole gel lanes were cut in 12-15 bands, proteins were in-gel tryptic digested and were subjected for mass-spectrometric analysis. Along with PKC-R (Figure 3) and GSK3-β/GSK3-R (Figures 2 and 3), we could specifically identify previously known targets of Bis-III in samples incubated with FLAG-coupled-Bis-III. These include identification of calcium/calmodulin dependent protein kinase II-delta, gamma (CaMKII ∆, γ), adenosine kinase, cell division protein kinase 2 (CDK2) and ribosyldihydronicotinamide dehydrogenase (NQO2). We also identified some unreported targets of Bis-III which include cAMPdependent protein kinase type I-alpha (PKAC-R), prohibitin, Journal of Proteome Research • Vol. 7, No. 8, 2008 3493
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Journal of Proteome Research • Vol. 7, No. 8, 2008 no. distinct peptides top matched peptides
Heat Shock Protein 90 Filamin A alpha STE20-like Serine/Threonine Protein Kinase (SLK) Peroxiredoxin-1 Ras GTPase Activating-like Protein IQGAP1 Perixiredoxin-2 Pyridoxal Kinase Peptidyl-prolyl cis-trans isomerase A Hormerin Heat Shock Protein 86 14-3-3 Protein theta Nucleoside Diphosphate Kinase B Nucleoside Diphosphate Kinase A poly(rC)-binding Protein 2 Rho-related GTP-Binding Protein RhoC Voltage Dependent Anion Selective Channel Protein 4 (VDAC4)
IPI00334775.5 IPI00302592.2 IPI00022827.1 IPI00000874.1 IPI00009342.1 IPI00027350.2 IPI00013004.1 IPI00419585.8 IPI00398625.5 IPI00031523.3 IPI00018146.1 IPI00026260.1 IPI00012048.1 IPI00012066.1 IPI00027434.1 IPI00084625.2
Resin-Bis-III Specific 30 GFEVVYMTEPIDEYCVQQLK 25 IPEISIQDMTAQVTSPSGK 22 DTILQTVDLVSQETGEK 18 HGEVCPAGWKPGSDTIKPDVQK 17 EELQSGVDAANSAAQQYQR 14 KEGGLGPLNIPLLADVTR 12 GQVLNSDELQELYEGLR 8 HTGPGILSMANAGPNTNGSQFFICTAK 8 GEQHGSSSGSSSSYGQHGSGSR 8 HNDOEQYAWESSAGGSF 7 AVTEQGAELSNEER 7 GDFCIQVGR 6 FMQASEDLLK 4 ESTGAQVQVAGDMLPNSTER 4 SIDSPDSLENIPEK 2 LTLSALLDGK
Specific IVVQGEPGDEFFIILEGSAAVLQR IYLTADNLVLNLQDESFTR DVIATDKEDVAFK DPEAPIFQVADYGIVADLFK MLVDDIGDVTITNDGATILK GIGMGMTVPISFAVFPNEDGSLQK FMDASALTGIPLPLIK
e0.000 e0.000 e0.000 e0.000 e0.000 e0.000 e0.002 e0.000 e0.000 e0.000 e0.000 e0.000 e0.000 e0.009 e0.000 e0.000
e0.000 e0.000 e0.000 e0.000 e0.000 e0.000 e0.000
e0.000 e0.000 e0.000
e0.000
e0.000 e0.000 e0.000 e0.000
peptide p-value12
e0.000 e0.000 e0.000 e0.002 e0.000 e0.002 e0.008 e0.000 e0.000 e0.000 e0.000 e0.000 e0.000 e0.011 e0.000 e0.005
e0.000 e0.000 e0.000 e0.000 e0.000 e0.000 e0.000
e0.000 e0.000 e0.000
e0.000
e0.000 e0.000 e0.000 e0.000
peptide q-value12
5.51 5.72 5.85 5.23 6.12 5.46 5.16 4.29 4.04 5.08 4.97 2.93 3.57 4.59 3.68 3.57
6.09 5.49 4.43 6.54 4.50 3.16 4.52
6.02 5.03 5.16
5.45
5.31 5.51 6.07 5.59
Sequest Xc
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.310 0.087 0.000 0.006 0.002
0.000 0.000 0.002 0.000 0.000 0.021 0.000
0.000 0.000 0.000
0.000
0.000 0.000 0.000 0.005
tandem e value
a Proteins identified using mass spectrometry where bait compound Bis-III is either FLAG-coupled or immobilized on resins. Please refer to the Supporting Information section 1 and Higgs et al.12 for the definition of ‘p-value’ and ‘q-value’. PKC-R is not listed since in all the samples it was identified using Western blot analysis (Figure 3).
cAMP-dependent Protein Kinase type I alpha (PKAC-R) Prohibitin-2 Malate Dehydrogenase Electron Transfers Flavoprotein Subunit alpha T-complex Protein 1 Subunit alpha Heme Binding Protein 1 Cell Division Protein Kinase 2 (CDK2)
FLAG-Coupled-Bis-III 16 16 9 8 8 6 5
FLAG-Coupled-Bis-III and Resin-Bis-III Ribosyldihydronicotinamide Dehydrogenase (NQO2) 30 VLCQGFAFDIPGFYDSGLLQGK Adenosine Kinase (AK) 20 VMPYVDILFGNETEAATFAR Prohibitin 12 AAELIANSLATAGDGLIELR Calcium/calmodulin-dependent Protein Kinase II delta (CaMKII 10 DLVTGGELFEDIVAR ∆) Calcium/calmodulin-dependent Protein Kinase II gamma 9 ITEQLIEAINNGDFEAYTK (CaMKII γ) Glycogen Synthase Kinase-3 beta (GSK3β) 6 IQAAASTPTNATAASDANTGDR Voltage Dependent Anion Selective Channel Protein 2 (VDAC-2) 5 SCSGVEFSTSGSSNTDTGK Voltage Dependent Anion Selective Channel Protein 1 (VDAC-1) 7 TDEFQLHTNVNDGTEFGGSIYQK
annotation
IPI00021831.1 IPI00027252.6 IPI00291005.7 IPI00010810.1 IPI00290566.1 IPI00148063.1 IPI00031681.1
IPI00028570.2 IPI00024145.1 IPI00216308.4
IPI00172450.2
IPI00219129.8 IPI00234368.1 IPI00017334.1 IPI00172636.3
protein ID
Table 1. Protein Identified Using Two Techniquesa
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Immuno-Chemo-Proteomics For Target Deconvolution voltage dependent anion-selective channel protein (VDAC) and heme binding protein which were consistently present in all three replicates (Table 1). PKAC-R and heme binding protein both contain a nucleotide binding site and Bis-III is a known ATP competitive inhibitor; hence, Bis-III interaction with these proteins can be explained. An earlier report where Bis-III was immobilized over solid support did not capture CDK2 in the pull-down experiment.8 However, in an in vitro experiment, Bis-III was shown to inhibit CDK2 activity with an IC50 value of ∼2 µm.8 It is possible that due to lower structural strain, FLAG-coupled-Bis-III was able to capture a lower affinity binding protein from the protein matrix. In a single experiment, we observed the presence of CDK3 and CDK4 in FLAG-coupledBis-III samples. Activities of these cell division protein kinases are inhibited by Bis-III but with lower affinity.9 Out of all the other eluted proteins, identification of VDAC was unexpected. VDAC is a major constituent of the outer mitochondrial membrane where it constitutes the pore-forming protein ‘porin’.18 VDAC interacts with kinases 19,20 and it is possible that VDAC was pulled out as a part of a complex. However, none of the known VDAC interacting kinases were identified. Other studies have shown that the protein kinase C inhibitor Ro 31-8220 inhibits voltage dependent sodium (anion) channels21 suggesting that FLAG-coupled-Bis-III may directly interact with the VDAC protein. Comparison of Target Binding Using Immobilized Bis-III on Solid Support to FLAG-Coupled-Bis-III. Cell lysate was incubated with immobilized Bis-III-matrix and bound proteins were eluted using 1 mM free Bis-III. Proteins were separated over SDS-PAGE and visualized using silver stain. Capture of the known target proteins PKC-R and GSK3-β was confirmed by Western blot analysis (Figure 3). Proteins isolated with FLAGcoupled-Bis-III were also run on a parallel gel and proteins were identified using mass spectrometry. We identified most of the proteins previously reported by Daub et al. 8 using the solid support method with Bis-III. Consistently in all the repeated three experiments, we observed the presence of CaMKII-∆, γ, SLK, NQO2, GSK3- β and Adenosine kinase. Additionally, we found that pyridoxal kinase, VDAC, prohibitin, nucleoside diphosphate kinase A and B, hydroxyacyl-Coenzymme A dehydrogenase type II, Ras GTPase-activating-like protein (IQGAP1) were also consistently present in all the three experiments. Table 1 lists all the identified proteins. Both techniques captured several of the same proteins including PKC-R, GSK3Ra/β, CaMKII-∆, γ, NQO2, VDAC and prohibitin. Pyridoxal kinase, SLK and IQGAP1 were identified only with Bis-III immobilized on the solid support not through FLAG-coupledBis-III. At the same time, PKAC-R, CDK2 and heme binding protein were specifically found through FLAG-coupled-Bis-III approach. Bis-III efficiently inhibits the activities of PKC-R, GSK3- β and NQO2.8,17 Bis-III also inhibits the activity of rCaMKII (species-rat) with an IC50 values of 0.86-5.3 µm (unpublished data). Although inhibition activity values can not be directly converted to binding affinity values, higher inhibition activity should correlate with higher binding affinity of the small molecule ligand. Thus, we hypothesize that the higher binding affinity proteins were captured using both the techniques. Additionally, high-abundance protein with lower affinity may also have been captured. However, the different coupling methods of Bis-III captured different proteins of lower binding affinity (Figure 4), perhaps due to differences in steric inhibition.
Figure 4. Comparative protein identification using two techniques. The modified Ven diagram shows the comparative number of proteins identified with particular technique. ‘N’ is the number of protein identified with a cutoff binding specificity ratio >0.8 (see Strategies for Target Deconvolution). Decreasing binding arrows represent the hypothesis that higher affinity binding proteins with Bis-III or higher abundance proteins were captured using both techniques; however, lower affinity binders showed steric specificity in binding to either one or other probe.
Strategies for Target Deconvolution. One of the major drawbacks to chemo-proteomic strategies is the presence of high levels of nonspecific protein binding. To identify proteins specifically interacting with Bis-III, we designed the following strategy (Figure 5). All experiments were repeated three times under identical conditions. Proteins identified with at least two distinct peptides having identification q-values 0.8 reduced the list of proteins to 1/50 of the original at the final stage of deconvolution.
Discussion The use of immobilized small molecules as affinity reagents for the identification of drug targets is gaining popularity. Journal of Proteome Research • Vol. 7, No. 8, 2008 3495
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Figure 5. Target deconvolution strategy. The specificity of the identified proteins was evaluated with the calculation of the binding specificity ratio (see text). Proteins identified with a binding specificity ratio
