Capture of Drug Targets from Live Cells Using a Multipurpose Immuno

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Capture of Drug Targets from Live Cells Using a Multipurpose Immuno-Chemo-Proteomics Tool Chaitanya Saxena,* Tabetha M. Bonacci, Karen L. Huss, Laura J. Bloem, Richard E. Higgs, and John E. Hale* Integrative Biology, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 Received March 25, 2009

Recently we have described the development of an Immuno-chemo-proteomics method for drug target deconvolution and profiling the toxicity of known drugs (Saxena, C.; Zhen, E.; Higgs, R. E.; Hale, J. E. J. Proteome Res. 2008, 8, 3490-3497). The orthogonal nature and advantage of the newly developed method over existing ones were presented. Most commonly, a small molecule was coupled to an epitope and used as an affinity probe to bind targets and later antibody against the epitope was used to isolate the probe-protein complex. However, such studies performed using cell lysates are prone to false positive identification because the protein source is not in its native physiological condition. Here we describe the development and application of a multipurpose soluble probe where a small molecule was coupled to a fluorophore-tagged cell-permeable peptide epitope, which was used to affinity isolate binding proteins from live cells. Fluorophore coupling allowed direct visualization of the compound in the cells, and cell permeability of the probe provided opportunity to capture the targets from the live cell. The GSK3-β inhibitor Bisindolylmaleimide-III was coupled to a peptide containing the fluoresceintagged TAT epitope. Following incubation with the live cells, the compound and associated proteins were affinity isolated using antifluorescein antibody beads. Using this approach, we captured the known Bisindolylmaleimide-III target GSK3-β and previously unidentified targets from live cells. Dose-dependent inhibition of target binding to probe in the presence of uncoupled compound validated the approach. This method was directly compared with the one where cell lysate was used as the protein source providing an advanced strategy to aid in target deconvolution and help to eliminate false positives originating from non-native protein source. Keywords: chemical proteomics • Bisindolylmaleimide III • GSK3-beta inhibitor • drug target deconvolution • cell-permeable peptide-coupled small molecule • mass spectrometry • affinity chromatography

Introduction To gain system-wide understanding of proteins that interact with small-molecule inhibitors, researchers need specific tools that can work with the physiologically relevant intact biological systems. Development of such specific tools is also very important from a drug discovery perspective, where failure rates of developing new drugs are very high. An early prediction of small-molecule inhibitor interaction with the physiologically relevant proteome can help de-risk the new candidate in early stages of development. These specific tools can also help in elucidating the biological mechanism of disease, mechanism of drug action, rational drug design, and patient stratification.1 In pursuit of developing these specific tools, recently we have described development of an immuno-chemo-proteomics method for target deconvolution where a peptide-coupledsmall molecule was used to identify the interacting proteome in the cell lysate.1 It was established that this immuno-chemo* To whom correspondence should be addressed. Chaitanya Saxena, Telephone Number: 317-651-1539. Fax Number: 317-276-9574. E-mail address: [email protected]. John E. Hale, Telephone Number: 317277-5373. Fax Number: 317-276-9574. E-mail address: [email protected]. 10.1021/pr900277x CCC: $40.75

 2009 American Chemical Society

proteomics method affords the ability to verify proteins identified through the solid-phase method but also to identify protein interactions that may be impaired by the solid phase geometry. Immuno-chemo-proteomics method provides two clear advantages over the existing chemical proteomics methods: (a) diffusion limited interaction of the peptide-coupled smallmolecule probe allows enhanced rates of association with the target protein and (b) binding characteristics of the probe with the target are directly verifiable. However, this immuno-chemoproteomics method and other chemical proteomics methods2-4 where the small molecules are immobilized on a solid support remain limited with the use of cell lysate as the protein-source, where physiological environment of protein has completely been disrupted. The process of cell-lysis might activate stress proteins which in-turn changes the proteome of the cell. Once the cells are ruptured, native protein-protein, proteinmetabolite and other biomolecular interactions might suffer leading to structural and functional changes in protein complexes. Protein-small-molecule interaction studies performed in these non-native, unphysiological conditions may not identify the true interacting partners and might increase the possibilities of false-positives identification. Journal of Proteome Research 2009, 8, 3951–3957 3951 Published on Web 06/22/2009

research articles To circumvent the shortcomings associated with using celllysates as a protein source, we developed a generalized strategy for target deconvolution from live cells. Here we describe the development and application of an immuno-chemo-proteomics technique for target deconvolution from live cells where the test molecule Bisindolylmaleimide III (Bis-III), a derivative of a known Protein Kinase C (PKC) and GSK3-β inhibitor,5-7 was coupled to a cell-permeable TAT peptide. The TAT tagged kinase inhibitor was demonstrated to retain inhibitory activity. The fluorophore coupled to this novel TAT-small-molecule construct confirms the delivery of the small molecule to the cells. Once the small-molecule construct interacted with the cells in its physiological conditions, inhibitor-protein complexes were isolated with an antifluorophore affinity resin. In comparison, the same small-molecule construct was incubated with the cell lysate and inhibitor-protein complexes were isolated similarly. We provide a comparative analysis of both techniques. The development of the cell-permeable peptidecoupled small-molecule approach provided us an opportunity to deliver the small molecule to the live cells and identify its interacting partners in physiological conditions. Cell permeability of the peptide provides an opportunity that this novel construct can be used in development of a cell-based assay for small-molecule inhibitor screening.

Material and Methods Reagents. Cell culture media was purchased from Invitrogen (Carlsbad, CA). Modified 5-FAM-TAT peptide with an extra cysteine residue at the c-terminal end (5-FAMYGRKKRRQRRRC-COOH) was custom synthesized at Anaspec Inc. (San Jose, CA). A heterobifunctional cross-linker 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). Mouse monoclonal anti-Glycogen Synthase Kinase 3 R/β (GSK3 R/β) antibody was purchased from Abcam (Cambridge, MA). BioMag Sheep Anti-Fluorescein magnetic beads were purchased from Qiagen (Valencia, CA). Preparation of 5-FAM-TAT-Coupled-Bis-III. Stock solutions of 13 mM Bis-III and LC-SMCC were prepared in DMSO. Final concentrations of 5 mM Bis-III and 5 mM LC-SMCC were added to DMSO in a 300 µL preparation. The reactant solution was kept under constant orbital mixing for 45 min in dark at 37 °C. From stock solution of 13 mM, 100 µL of modified 5-FAM-TAT peptide (5-FAM - YGRKKRRQRRRC-COOH) was added to the solution and mixing was continued for another 45 min in dark at 37 °C. The resultant solution was subjected to gel-filtration using Sephadex G-25 (Sigma) to remove unreacted Bis-III and LC-SMCC. Gel-filtration flow through containing unreacted 5-FAM-TAT peptide, 5-FAM -TAT reacted to LC-SMCC and 5-FAM -TAT coupled to Bis-III through LCSMCC 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 30 min with a flow rate of 0.5 mL/min using an Agilent 1100 HPLC pump. Fractions were collected every one minute and were analyzed using a MALDITOF (4800 Proteomic analyzer, Applied Biosystems). Fractions showing peaks at m/z 2739.3 were pooled together and lyophilized. The concentration of thus obtained “5-FAM -TAT3952

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Saxena et al. coupled-Bis-III” was measured by acquiring absorption spectra of the 5-FAM at 490 nm and of the Bis-III at 540 nm. Cell Culture, Probe Incubation, Microscopy, and Cell Lysis. HEK cells were cultured in poly-lysine coated six-well plate. Semiconfluent cultured cells were washed twice with HBSS. Cells were incubated at 37 °C for 10 min with 40 µM of 5-FAM-TAT-coupled-BisIII or 5-FAM-TAT-linker (control probe) solution prepared in HBSS. Excess probe solution was removed and cells were washed three times with HBSS, five times with 0.2 N acetic acid/150 mM NaCl (pH 2.5) to remove the probe which might be hydrophobically bound to the cell membrane. Cells were finally returned to HBSS. Cells images were acquired immediately to avoid any potential cell death due to excess probe concentration containing Bis-III. Fluorescence images were acquired by exciting the 5-FAM in the range of 470 nanometer- 500 nanometer. For target capture experiments confluent cultured cells were washed twice with PBS. Cells were incubated at 37 °C for 10 min with 40 µM of 5-FAM-TATcoupled-BisIII or 5-FAM-TAT-linker (control probe) solution prepared in PBS. Excess probe solution was removed and cells were further washed twice with PBS. Cells were scrapped and gently lysed in lysis 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 and 1 mM DTT and protease inhibitor cocktail. Concentration of the probe in the lysate was determined by monitoring absorption of fluorescein at 490 nm. Lysate was centrifuged at 800 g for 10 min at 4 °C to remove the cellular debris. Pellets were discarded and supernatant was collected for further use. In the “cell-lysate” experiment, cells were not incubated with the probe. The cells were scrapped and gently lysed in buffer. Lysate thus obtained was centrifuged at 800 g for 10 min at 4 °C to remove the cellular debris. Pellets were discarded and supernatant was collected. And this stage, lysate was incubated with final concentration of 85 µM 5-FAM-TAT-coupled-BisIII or 5-FAM-TAT-linker (control probe) (Please see supplement 1 for the calculation of final probe concentration, Supporting Information). Affinity Chromatography with 5-FAM-TAT-coupledBis-III. Supernatant obtained after centrifugation was incubated with optimized (supplement 2) 2.5 mL BioMag Sheep Anti-Fluorescein magnetic bead suspension for 45 min at 4 °C. Flow through was removed and beads were washed three times using 1000 µL of the lysis buffer (without 10 mM sodium pyrophosphate and 1 mM DTT and protease inhibitor cocktail) each time. The beads were eluted three times with 200 µL of elution buffer containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA and 1 mM of Bis-III each time. Due to the poor solubility of Bis-III in aqueous solution, a stock solution of BisIII was prepared in DMSO and the required amount of Bis-III was diluted in the buffer to prepare the elution buffers: thus the elution buffer contained less than 1% of DMSO. Cell-Based Inhibition of GSK3-β Binding to the 5FAM-TAT-coupled-Bis-III Probe. HEK cells were cultured to confluence in a 24-well plate. Culture medium was removed and cells were incubated with 300 µL of varying concentration of Bis-III dissolved in culture medium. After 1 h of incubation, at 37 °C, Bis-III solution was removed from the wells and cells were washed in following way: 1 mL of HBSS was added to the cells and cells were kept in this solution for five minute at room temperature. HBSS was then removed from the wells. The process was repeated for three times and the washes were collected every time. The amount of free Bis-III present in the

Immuno-Chemo-Proteomics for Target Capture From Live Cells washes was monitored using absorption spectrophotometer. After the third wash no Bis-III was detected in the wash solution. After the removal of the third HBSS wash, 300 µL of 10 µM solution of 5-FAM-TAT-coupled-Bis-III prepared in HBSS was added to the wells and cells were incubated at 37 °C for 30 min. Later, solution was removed from the well and cells were washed once with HBSS. At this stage 1 mL of lysis 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 was added to the wells, cells were scrapped and cell lysis was prepared by gentle pipetting of the cells. One-hundred microliters of this lysate were taken out from each well for relative protein concentration estimation and the rest 900 µL was used for the 5-FAM-TAT-coupled-Bis-III and associated protein complex capture. Cell-lysate was taken out from wells and added to an eppendorf tube containing 2.5 mL of prewashed and drained anti-FITC magnetic beads. This cell-lysateantibody mixture was incubated at 4 °C for 45 min. Later, using a magnetic bead separator flow through was removed and beads were washed two times using 1000 µL of the lysis buffer (without 10 mM sodium pyrophosphate, 1 mM DTT and protease inhibitor cocktail) each time. 100 µL of LDS sample buffer with reducing agent was added to beads and beads were placed at 70 °C for 10 min. Magnetic beads were removed and proteins from 30 µL of this solution were separated over 4-12% SDS/PAGE gel. Proteins were transferred to a nitrocellulose membrane and immunoblotted with the GSK3-β antibody. For data-analysis, protein concentrations from different samples were normalized prior to immunoblot analysis. Protein Sample Preparation for Analysis. Eluted proteins were concentrated in a speedvac system and were subsequently precipitated using a chloroform-methanol precipitation method.8 Precipitated proteins were dissolved in the LDS sample buffer (Invitrogen), separated using 4-12% SDS/PAGE gel and were visualized using Coomassie blue 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.9 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 reversephase column and eluted with a gradient of 5 to 50% CH3CN developed over 60 min. 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.10 Details of the protein identification process are attached in supplementary method (section 3 and 4, Supporting Information). For the final set of identified proteins, a protein deconvolution strategy was adopted as described earlier1 with minor modifications (supplement section 4, Supporting Information). GSK3-β Filter Binding Assay. The kinase reactions for the GSK3-β filter binding assay IC50 determinations consisted of 50 mM MOPS, pH 7.0, 0.03% Triton X-100, 12.5 mM MgCl2, 4% DMSO, 50 µM pCREB peptide (KRREILSRRP(pS)YR), 50 µM (1*KM) ATP, human GSK3-β enzyme (Invitrogen), 0.5 µCi [γ33P] ATP (ICN) per well. Bis-III, 5-FAM-TAT-coupled-Bis-III and

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5-FAM-TAT-linker (control peptide) were dissolved at 10 mM in 100% DMSO and then diluted to 100 µM with 20% DMSO. Further serial dilutions (1:3) were prepared over a 100 µM to 0.005 µM range. Kinase reactions were performed in 96-well polystyrene plates. Reagents were added to the plate as follows: 5 µL 20% DMSO (“MAX”) or 500 mM EDTA (“MIN”) or inhibitor, 15 µL ATP and pCREB peptide, and 5 µL enzyme. Eight IC50 determinations and 16 controls (eight uninhibited “MAX” and eight 500 mM EDTA inhibited “MIN” control wells) were performed per 96-well plate. Kinase reactions were incubated at room temperature for 60 min (this reaction time had been determined experimentally to be within initial velocity kinetics of the assay) and then the reactions were quenched by the addition of 100 µL of 10% H3PO4 and then 100 µL of the each quenched reaction was transferred to a well of a 96-well phosphocellulose membrane filter plate (Millipore). The plate was filtered, washed with 3 volumes of 0.5% H3PO4, and then vacuum-dried. After adding the scintillation cocktail the plates were read by scintillation counting. Absolute compound IC50 values were determined using CPM (counts per minute) values by calculating percent inhibition with respect to on-plate “MIN” and “MAX” controls and then fitting the percent inhibition values and 10-point dose response data to a fourparameter logistic equation using ActivityBase (ver.4.1, IDBS).

Results Development and Characterization of a Multipurpose Peptide-Coupled Small-Molecule Probe. In an attempt to develop a multipurpose probe to visualize and capture the cellular targets in live-cell conditions, we sought an affinity handle to satisfy three conditions, (a) should carry the small molecule inside the cell in a freely diffusable form, (b) can be easily located once inside the cell, and (c) should be easily isolatable once the small molecule has interacted with the cellular proteins. HIV-TAT, a 9 amino acid peptide (NH2GRKKRRQRRR-COOH) derived from the HIV-1 Tat protein, has been successfully shown to deliver a large variety of cargos including nucleic acids, peptides, and proteins to the cell.11-14 Usually cargos for cellular delivery are attached onto N- or Cterminal of the peptide. We selected TAT-peptide as the “handle” to be attached to Bis-III for cellular delivery. A modified form of the TAT-peptide where a fluorescein (5-FAM) added to its N-terminus end and cysteine residue added to its C-terminus (5-FAM-YGRKKRRQRRRC-COOH) 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 5-FAM-TAT-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 shows the coupling scheme of the modified TAT-peptide with Bis-III. To reduce the reaction of the primary amine of the modified TAT-peptide with the NHS moiety of the LC-SMCC, Bis-III was reacted first with LC-SMCC and the modified TAT-peptide was added later in the reaction (see material and methods). 5-FAM-TATcoupled-Bis-III was purified by gel-filtration chromatography followed by RP-HPLC. Coupling was characterized by acquiring the mass spectra of the purified 5-FAM-TAT-coupled-Bis-III which displayed an m/z value at 2739.3 (Figure 1B), which is equal to theoretical m/z value of the probe. To ascertain that 5-FAM-TAT-coupled-Bis-III is still an active kinase inhibitor, a kinase activity inhibition assay of GSK3-β was performed in the presence of free Bis-III or 5-FAM-TAT-coupled-Bis-III since Journal of Proteome Research • Vol. 8, No. 8, 2009 3953

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Figure 1. Preparation and characterization of 5-FAM-TAT-coupledBis-III probe. (A) Structure of GSK3-β inhibitor Bis-III and the linker (LC-SMCC) used to couple Bis-III with the modified 5-FAM-TAT peptide. (B) Mass spectrum of the purified 5-FAM-TAT-coupledBis-III showing m/z peak at 2739.3. (C) GSK3-β kinase activity inhibition assay in presence of Bis-III and 5-FAM-TAT-coupledBis-III. (D) Fluorescence Image of the HEK cells showing that probe does enter the cell.

GSK3-β is a known target of Bis-III.5-7 Free Bis-III inhibited GSK3-β with an IC50 value of ∼0.14 µM. The 5-FAM-TATcoupled-Bis-III inhibited GSK3-β with an IC50 value of ∼0.25 µM (Figure 1C). Because 5-FAM-TAT-coupled-Bis-III showed approximately similar kinase inhibition efficiency, we concluded that the interaction of 5-FAM-TAT-coupled-Bis-III with 3954

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Saxena et al. its binding partner would be retained when the probe enters in the cells and interacts with the cellular proteins. Optimization of Cellular Entry of the 5-FAM-TATcoupled-Bis-III Probe. The assumption that TAT-peptidecoupled Bis-III will enter the cell was confirmed by incubating the 40 µM of 5-FAM-TAT-coupled-Bis-III probe or control probe, dissolved in HBSS, with the HEK cells. After 10 min incubation at 37 °C, excess probe was removed and cells were acid washed to remove the probe which might be hydrophobically bound to the cell membrane. Acquired fluorescence image (Figure 1D) confirmed that probe did enter the cell. In the experiment where control probe 5-FAM-TAT-linker was incubated with the cell showed much higher fluorescence when compared to those where 5-FAM-TAT-coupled-Bis-III probe was used. Coupled Bis-III in the probe provides a possibility of fluorescence resonance energy transfer because emission spectrum of 5-FAM overlaps with the lower energy absorption spectrum of the Bis-III which extends up-until 550 nm (supplement section 5, Supporting Information). Interestingly when images were monitored under red-channel (λ > 580 nm) a very low intensity emission from the cells, probably from the BisIII emission, was observed (data not shown) when 5-FAM-TATcoupled-Bis-III probe was used and not when the control probe was used. This further confirmed the delivery of 5-FAM-TATcoupled-Bis-III to the cells. Capture of Target Protein GSK3-β and Dose-Dependent Inhibition of Interaction in Presence of Free Inhibitor. The assumption that with 0.25 µM IC50 value 5-FAM-TAT-coupledBis-III will bind to its target proteins was confirmed by a probe pull-down experiment. GSK3-β in the live HEK cells specifically interacted with the probe and was enriched after anti-5-FAMTAT chromatography, while comparatively very low binding was visible in the control experiments where Bis-III was not conjugated to 5-FAM-TAT peptide (Figure 2). In optimizing affinity chromatographic conditions we found that the amount of antibody used with given protein concentration was critical in capturing 5-FAM-TAT-coupled-Bis-III probe. We used the optical absorption of 5-FAM to determine the concentration of probe at various stages of the experiment. Probe was incubated with antibody for 45 min or overnight, however there was no significant difference in the amount of probe captured by the antibody under these two different conditions. To ascertain the specificity of capturing the target protein by 5-FAM-TAT-coupled-Bis-III probe, cells were pre incubated with free Bis-III at different concentrations and a single concentration of probe was used to measure the decrease in capture of the GSK3-β from the cells. As expected increasing concentration of free-Bis-III in cells inhibits the binding of GSK3-β to the probe and thus the amount of GSK3-β captured with similar concentration of probe was decreased in westernblot analysis (Figure 3A). To conclude that this decrease in binding indeed is dose-dependent, a linear kinetic interaction range of antifluorescein antibody with the probe was optimized (data not shown). In the given experimental conditions, as described in materials and methods, a maximum of >56% reduction in GSK3-β binding to probe was observed. A four parameter fit provided a cell based EC50 value of 17.93 µM for the inhibition of GSK3-β binding to the probe in presence of free Bis-III. Although this value has no biological meaning, it can directly be used for inhibitor screening in drug discovery for the same protein target in defined experimental conditions. Once it was established that 5-FAM-TAT-coupled-Bis-III can capture the target protein GSK3-β from the live cell, we

Immuno-Chemo-Proteomics for Target Capture From Live Cells

Figure 2. Elution profile of proteins from anti-FITC antibody resins. (A) In three independent live cell experiments eluted proteins were concentrated before SDS-PAGE analysis and visualized using Coomassie blue stain. (B) Corresponding samples were analyzed with immunoblotting technique using a monoclonal antibody against GSK3-R/β protein. (C) Samples from celllysate experiment were analyzed with immunoblotting technique using a monoclonal antibody against GSK3-R/β protein. *n/a- not acquired.

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. Using Coomassie blue staining most of the proteins visualized on the gel appears to be present in both the Bis-III and control probe lanes and thus largely represent nonspecifically bound proteins. To identify the proteins whole gel lanes were cut in 8-10 bands, proteins were reduced, alkylated, in-gel trypsin digested and were subjected for mass-spectrometric analysis. To identify the proteins specifically captured in 5-FAM-TATcoupled-Bis-III experiment, a robust strategy involving repetition of the experiments, selecting commonly identified proteins and comparing them with the protein identified from the control experiment was adapted (supplement section 4, Supporting Information). To our surprise, we identified many unreported targets of Bis-III which include ATP-dependent RNA helicases (DDX1, DDX5 and DDX9), heterogeneous nuclear ribonucleoproteins (hRNPs U, Q, A0), t-RNA synthetases (Leucyl and Lysyl), Eukaryotic translation initiation factor 5A-2 (ElF5A2) and many other proteins (Table 1). GSK3- β was identified with two peptides in one out of three experiments and with one peptide in the other two experiments. Thus, we list the peptide which was identified in all the experiments following our selection criteria (supplement section 4, Supporting Information) that protein should be identified in all the three experiments. The MS/MS spectra of the GSK3- β peptide that was seen in all 3 experiments is shown in supplemental figure S3, Supporting Information. Out of 25 proteins identified, 16 proteins contain a nucleotide binding site. Since Bis-III is a known ATP competitive inhibitor, the interaction of the probe

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Figure 3. Western-blot analysis of GSK3-R/β bound to the probe after incubating the cells with varying concentration of Bis-III. (A) Increasing concentration of free-Bis-III in cells inhibits the binding of GSK3-β to the probe and thus amount of GSK3-β captured to similar concentration of probe was decreased in western-blot analysis. (B) Inhibition of GSK3-R/β binding to the probe was found to be dose-dependent. Inset shows only the exponents for the entire concentration range of the Bis-III. Average data from three independent experiments are presented.

with these proteins can be mediated by this potential binding site. How the other identified 9 proteins interact with the probe is not clear. It could be that they have some unknown Bis-III interacting site or they were pulled out as a part of a complex. Comparison of Target Binding Using Cell Lysate As Protein Source. Cell lysate prepared from the HEK cells was incubated with 85 µM 5-FAM-TAT-coupled-Bis-III (supplement 1 describes the rational for the concentration used here, Supporting Information). After the affinity chromatography, proteins bound to the antibody beads were eluted using 1 mM free Bis-III. Proteins were separated over SDS-PAGE and the capture of the known target protein GSK3-β was confirmed by western-blot analysis (Figure 2). GSK3-β in the cell-lysate specifically interacted with the probe and was enriched after anti-5-FAM-TAT chromatography, while no binding was visible in the control experiments where Bis-III was not conjugated to 5-FAM-TAT peptide. From western-blot analysis it appears that more GSK3- β was captured in the cell-lysate experiment; however, because the two Western blots shown in Figure 2 were run at different times, no comparative quantitative information on the amount of target capture was obtained. Proteins isolated with 5-FAM-TAT-coupled-Bis-III were also run on a parallel gel and proteins were identified using mass spectrometry. Consistently in all the repeated three experiments, we observed the presence of heterogeneous nuclear ribonucleoproteins (hRNPs A1, A3), Eukaryotic translation initiation factor 5A-2 (ElF5A2), ATP synthase and many other proteins (Table 1). Additionally, we found that Nucleoside diphosphate kinase A, heat-shock protein 60, T-Complex protein subunit beta and theta were also consistently present in all the three experiJournal of Proteome Research • Vol. 8, No. 8, 2009 3955

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Saxena et al. a

Table 1. Protein Identified Using Live Cells and Cell Lysate

protein ID

annotation

no. distinct peptides

top matched peptide

IPI00030910.1 IPI00008612.2 IPI00300408.3 IPI00014238.2 IPI00386491.5 IPI00019912.2 IPI00453473.5 IPI00103525.1 IPI00011913.1 IPI00016621.6 IPI00017617.1 IPI00031683.1 IPI00018140.3 IPI00219622.2 IPI00028004.2

Proteins identified from live cell Glycogen synthase kinase-3 beta 1 DTPALFNFTTQELSSNPPLATILIPPHAR Eukaryotic translation initiation factor 5A-2 5 VHLVGIDIFTGK ATP-dependent RNA helicase DDX9 22 PSAAGINLMIGSTR Leucyl-tRNA synthetase, cytoplasmic 15 LALADAGDTVEDANFVEAMADAGILR ATP-dependent RNA helicase DDX1 9 FLVLDEADGLLSQGYSDFINR Exosome complex exonuclease RRP44 8 SLAESLDQAESPTFPYLNTLLR THO complex subunit 4 7 PMNIQLVTSQIDAQR Poly(A)-binding protein 4 7 SLGYAYVNFQQPADAER HSPC117 protein 7 GLGHQVATDALVAMEK Insulin-like growth factor 2 mRNA 5 SSFMQAPEQEMVQVFIPAQAVGAIIGK binding protein 1 Caprin-1 5 QGLNGVPILSEEELSLLDEFYK 38 kDa protein (Peptidase 39 M family) 3 AYDGTTYLPGIVGLNNIK Copper homeostasis protein cutC homologue 3 LYGADGLVFGALTEDGHIDK Lysyl-tRNA synthetase 3 YLDLILNDFVR Heterogeneous nuclear ribonucleoprotein U 2 EKPYFPIPEEYTFIQNVPLEDR Peroxisomal multifunctional enzyme type 2 2 VAVAIPNRPPDAVLTDTTSLNQAALYR Histone H4 2 TVTAMDVVYALK paraspeckle protein 1 2 NLSPVVSNELLEQAFSQFGPVEK Heterogeneous nuclear ribonucleoprotein A0 2 LFIGGLNVQTSESGLR AP-2 complex subunit alpha-2 2 IIGFGSALLEEVDPNPANFVGAGIIHTK Probable ATP-dependent RNA helicase DDX5 1 APILIATDVASR Short transient receptor potential channel 6 1 IVEAILSHPAFAEGKR Heterogeneous nuclear ribonucleoprotein Q 1 DLFEDELVPLFEK Proteasome subunit alpha type-2 1 YNEDLELEDAIHTAILTLK Proteasome subunit beta type-3 1 LYIGLAGLATDVQTVAQR

IPI00006935.2 IPI00472102.3 IPI00007765.5 IPI00215965.1 IPI00303476.1 IPI00025252.1 IPI00302925.3 IPI00012048.1 IPI00297779.6 IPI00186290.5 IPI00298994.5 IPI00012066.1 IPI00076042.2 IPI00419373.1 IPI00028570.2

Proteins identified from cell lysate Eukaryotic translation initiation factor 5A-2 1 VHLVGIDIFTGK Heat shock protein 60 8 AAVEEGIVLGGGCALLR Stress-70 protein, mitochondrial (precursor) 8 AQFEGIVTDLIR Heterogeneous nuclear ribonucleoprotein A1 7 GFGFVTYATVEEVDAAMNAR ATP synthase subunit beta 6 AIAELGIYPAVDPLDSTSR Protein disulfide-isomerase A3 4 DLLIAYYDVDYEK T-complex protein 1 subunit theta 4 DMLEAGILDTYLGK Nucleoside diphosphate kinase A 3 GDFCIQVGR T-complex protein 1 subunit beta 3 LALVTGGEIASTFDHPELVK Elongation factor 2 2 ALLELQLEPEELYQTFQR 271 kDa protein (similar to Talin 1) 2 TLAESALQLLYTAK poly(rC) binding protein 2 isoform b 2 IITLAGPTNAIFK Short heat shock protein60 Hsp60s2 2 TALLDAAGVASLLTTAEVVVTEIPK Heterogeneous nuclear ribonucleoprotein A3 1 LFIGGLSFETTDDSLR Glycogen synthase kinase-3 beta*

IPI00028570.2 IPI00218084.5 IPI00215638.5 IPI00103994.4 IPI00293655.3 IPI00183462.7 IPI00328840.8 IPI00012726.4 IPI00550689.3 IPI00008557.3

peptide peptide tandem p q Sequest e value value Xc value

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

3.82 3.45 4.90 6.24 6.04 4.93 5.44 5.51 4.86 6.09

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.050 0.000

0.000 0.000 0.001 0.000 0.000 0.000 0.008 0.000 0.000 0.000 0.000 0.008 0.000 0.000 0.000

0.000 0.000 0.001 0.000 0.000 0.000 0.008 0.000 0.000 0.000 0.000 0.008 0.000 0.000 0.000

4.41 4.60 2.31 4.43 5.34 5.44 2.74 4.97 5.22 5.17 3.91 2.02 4.25 4.08 5.09

0.000 0.000 0.000 0.037 0.000 0.000 0.023 0.000 0.000 0.000 0.000 0.390 0.002 0.002 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

2.97 4.40 4.08 5.86 3.56 4.02 4.44 2.86 1.90 3.34 4.09 3.47 4.12 4.46

0.054 0.000 0.001 0.000 0.000 0.006 0.001 0.670 0.008 0.005 0.000 0.004 0.000 0.000

a Proteins identified using mass spectrometry where protein source was either from live cell or cell-lysate experiment. Please refer to the supplementary material section 3 (Supporting Information) and Higgs et al.10 for the definition of “p value” and “q value”. * protein was identified using immuno-blot technique.

ments. Table 1 lists all the identified proteins. Experiments performed either using live-cell or cell-lysate captured GSK3R/β, and Eukaryotic translation initiation factor 5A-2. ATP dependent RNA helicase DDX1, DDX5, and DDX9 were identified only when probe was incubated with the live cell but not when cell lysate was used as protein source. On the other hand Nucleoside diphosphate kinase A, heat-shock protein 60 and T-Complex protein subunits were specifically found when cell lysate was used as the protein source. Interestingly, in both live-cell and cell-lysate experiments, the probe captured the distinct proteins of similar heterogeneous nuclear ribonucleoproteins (hRNP) family. The live-cell experiment captured hRNPU, hRNPQ, and hRNPA0 but the cell-lysate experiment captured hRNPA1 and hRNPA3. In total, 10 out of 14 proteins identified using the cell lysate as the protein source contain a nucleotide binding site. When compared head to head by protein annotation, only two proteins were commonly identified in both experiments. 3956

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However, when we compared for the proteins which can bind a nucleotide (Bis-III is a competitive ATP inhibitor) in both the experiments, we found that overlap of proteins identified using both the techniques was increased significantly (Figure 4).

Discussion The use of peptide-coupled small molecules as affinity reagents for the identification of drug targets provides decent target deconvolution.1 However, the use of cell lysates as a protein source to perform these studies may increase the chances of false positive identification because of the perturbed physiologicalconditionsoftheproteins.Disruptedprotein-protein, protein-DNA, protein-lipid, and other biomolecular interactions in a cell lysate may limit the capture of the right protein target by the peptide-coupled small-molecule probe. In this study, we described the development of a novel technique for capturing cellular binding partners of a small-molecule kinase

research articles

Immuno-Chemo-Proteomics for Target Capture From Live Cells

Conclusions

Figure 4. Comparative protein identification using live cells and cell lysate. “N” is the number of protein identified with at least one distinct peptide with a specific binding cutoff ratio > 0.75 (see Supporting Information Section 4: Strategies for target deconvolution). (A) Venn-diagram shows the comparative number of proteins identified using live cell or cell-lysate experiments based on the protein annotation. (B) Identified proteins in both the experiments were compared based on the possibilities that protein can bind a nucleotide.

inhibitor from the live cells. This method affords the ability to capture the known target protein of the small molecule from the live cell and also allow possibilities of locating the probe in the cell. Although in this method disruption of the cells was necessary to capture the probe along with its binding partners, the possibility that probe would have interacted with its binding partners in the cell lysate was minimal because concentration of probe present in the cell-lysis was significantly low (1.1 µM). In a separate experiment, a 2.0 µM concentration of probe could not capture any specific proteins over the control experiment (data not shown). In small-molecule affinity chromatographic experiments, nonspecific binders and high abundance proteins tend to dominate the list of proteins identified15 and complicates the drug target deconvolution processes. We used a similar strategy as described earlier,1 but with one distinct peptide cutoff for identification compared to two distinct peptides cutoff, for our data analysis. It should be noted that the experiments described herein are primarily aimed at hypothesis generation and identified proteins should be verified with at least one orthogonal method to ensure their presence in the sample. Although only one peptide from GSK3-β was identified in all three replicate experiments, we confirmed its enrichment with the Bisindoylmaleimide probe over the control experiments using western-blot analysis. We repeatedly found that this strategy, where identification of protein is established in multiple replicates over control experiments, provides robust criteria for the identification of proteins which are specifically interacting with the small molecule. The cell-permeable peptide-coupled-small molecule strategy may offer several advantages. As demonstrated, in the presence of a competitive small-molecule inhibitor, the capture of the target protein to the probe, from the cells, decreased in a dosedependent manner. This affinity of the tagged molecule to known targets can directly be used for small-molecule inhibitor screening in a cell-based assay. Additionally this assay can also determine the cell penetrability of the test compound, because the competitive binding of the target protein to the probe or the small molecule presumably occurs inside the cell. Thus, this newly developed probe can be used for multiple purposes beyond identifying the target proteins of small molecules from live cells.

With the present work we successfully demonstrated the development of a novel tool to capture the intracellular targets of a small molecule from the live cell. A comparative analysis of proteins captured from the live cell and the cell-lysate was performed. It was demonstrated that both techniques efficiently capture the protein targets of the small molecule and a subset of the same class of proteins; however, additional captured proteins were found to be specific to the individual protein source selected perhaps due to different biochemical and physiological conditions of the proteins. Further, it was shown that cell-permeable peptide-coupled small-molecule probe can be evolved into a cell-based assay for screening small-molecule inhibitor for an intracellular target. We believe that presented advanced immuno-chemo-proteomics approach will become a useful tool for the future drug target deconvolution and smallmolecule inhibitor screening strategies.

Acknowledgment. We thank Michael D. Knierman and Zhaoyan Jin for their technical assistance during mass spectrometric data acquisition. We also thank Eugene Zhen and Jesus A. Gutierrez for their help during data discussion to improve the experiment design. Supporting Information Available: Section (1) Calculation of HEK cell volume and concentration of probe, Section (2) Antibody optimization, Section (3) Data Analysis, Section (4) Strategies of Target Deconvolution and table of Identified Proteins using mass spectrometry with the binding specificity ratios, and Section (5) UV-vis Absorption Spectrum of Bis-III, 5-FAM-TAT-Cys peptide, 5-FAM-TAT-coupled-BisIII. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Saxena, C.; Zhen, E.; Higgs, R. E.; Hale, J. E. J. Proteome Res. 2008, 8, 3490–3497. (2) Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blencke, S.; Gutbrod, H.; Salassidis, K.; Stein-Gerlach, M.; Missio, A.; Cotten, M.; Daub, H. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15434–15439. (3) Knockaert, M.; Gray, N.; Damiens, E.; Chang, Y. T.; Grellier, P.; Grant, K.; Fergusson, D.; Mottram, J.; Soete, M.; Dubremetz, J. F.; Le Roch, K.; Doerig, C.; Schultz, P.; Meijer, L. Chem. Biol. 2000, 7, 411–422. (4) Guiffant, D.; Tribouillard, D.; Gug, F.; Galons, H.; Meijer, L.; Blondel, M.; Bach, S. Biotechnol J. 2007, 2, 68–75. (5) Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-Perret, T.; Ajakane, M.; Baudet, V.; Boissin, P.; Boursier, E.; Loriolle, F.; Duhamelll, L.; Charon, D.; Kirilovsky, J. Biol. Chem. 1991, 266, 15771–15781. (6) Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Biochem. J. 2000, 351, 95–105. (7) Hers, I.; Tavare´, J. M.; Denton, R. M. FEBS Lett. 1999, 460, 433– 436. (8) Wessel, D.; Flu ¨ gge, U. I. Anal. Biochem. 1984, 138, 141–143. (9) Hale, J. E.; Butler, J. P.; Gelfanova, V.; You, J. S.; Knierman, M. D. Anal. Biochem. 2004, 333, 174–81. (10) Higgs, R. E.; Knierman, M. D.; Bonne Freeman, A.; Gelbert, L. M.; Patil, S. T.; Hale, J. E. J. Proteome Res. 2007, 6, 1758–1767. (11) Fawell, S.; Seery, J.; Daikh, Y.; Moore, C.; Chen, L. L.; Pepinsky, B.; Barsoum, J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 664–668. (12) Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010– 16017. (13) Brooks, H.; Lebleu, B.; Vives, E. Adv. Drug. Deliv. Rev. 2005, 4, 559– 77. (14) Lindsay, M. A. Curr. Opin. Pharmacol. 2002, 5, 587–594. (15) Mano, N.; Sato, K.; Goto, J. Anal. Chem. 2006, 78, 4668–4675.

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