Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach—Based on a Target Profiling Study of Andrographolide

CHAPTER FIFTEEN

Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach— Based on a Target Profiling Study of Andrographolide J. Wang*,†,{,§,1,2, Y.K. Wong†,{,1, J. Zhang†,||,1, Y.-M. Lee{, Z.-C. Hua*,2, H.-M. Shen†,¶,2, Q. Lin{,2 *The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, China † Yong Loo Lin School of Medicine, National University of Singapore, Singapore { Faculty of Science, National University of Singapore, Singapore § Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research & Technology (SMART), Singapore ¶ NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore jj Clinical Research Institute, Zhejiang Provincial People’s Hospital, Hangzhou, China 2 Corresponding authors: e-mail address: [email protected]; [email protected]; [email protected]; [email protected]

Contents 1. Introduction and General Principles 2. Materials, Equipment, and Solutions 2.1 Materials 2.2 Equipment 2.3 C18 Buffers (for Section 3.6) 2.4 Gradient Separation Buffer (for Section 3.7) 3. Protocol 3.1 Cell Culture and Probe Treatment 3.2 Click Chemistry Tagging With Biotin Alkyne 3.3 Streptavidin Affinity Purification 3.4 (iTRAQ) Labeling 3.5 Sample Clean up by Strong Cation Exchange Chromatography 3.6 Desalting of Labeled Samples by C18 Column 3.7 Nano-LC Electrospray Ionization MS 3.8 Protein Identification and Quantification Using ProteinPilot™ Software 4. Discussion Acknowledgments References 1

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These authors contributed equally to this work.

Methods in Enzymology, Volume 586 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2016.09.049

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2017 Elsevier Inc. All rights reserved.

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Abstract Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule–protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products. A modified drug probe with a click chemistry-enabling addition is synthesized and used in live cell treatments where it undergoes covalent interactions with its cognate cellular targets. The probes are then ligated to biotin through click chemistry and enriched with avidin beads, followed by iTRAQ labeling and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for protein identification and relative quantitation discriminating specific targets from nonspecific binding proteins. The presented protocol has been used to successfully profile prominent drugs and natural products including andrographolide, aspirin, curcumin, etc., and can be a powerful tool to study the molecular mechanisms of bioactive small molecules.

1. INTRODUCTION AND GENERAL PRINCIPLES Bioactive small molecules, from naturally derived products of traditional medicine to modern synthetic drugs, have been an integral part of medicine throughout human history (Carlson, 2010; Ziegler, Pries, Hedberg, & Waldmann, 2013). With the explosive growth of knowledge and understanding of functional genomics and the cell proteome, the demand for novel drugs to modulate these cellular targets is ever increasing. In parallel with target-based drug development, in which a drug is designed specifically for a desired target with a known function, the phenotypic (or “forward”) approach screens drugs or compounds for desirable functional effects at the cellular level, thereby ensuring that selected drugs of interest are already capable of eliciting the required response (Schenone, Dancˇ´ık, Wagner, & Clemons, 2013; Wright & Sieber, 2016). This approach, however, gives little information about the actual molecular targets of the screened compounds. Clearly, such an approach to drug design must be accompanied by the ability to identify these targets in a comprehensive and unbiased manner (Fonovi
c & Bogyo, 2008; Nomura, Dix, & Cravatt, 2010; Paulick & Bogyo, 2008; Speers & Cravatt, 2005). The importance of target identification is magnified when we consider the fact that drugs are often promiscuous, a property that is also commonly observed in naturally derived bioactive compounds (Hopkins, 2009; Schenone et al., 2013). Finally, it is desirable to fully understand a drug’s potential off-target effects and adverse reactions, and this can be greatly facilitated by an unbiased screen of its

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cellular targets (Schenone et al., 2013; Yang et al., 2010). Target identification to understand the full target spectrum of a drug is thus critical not only to appreciate and apply its therapeutic effects, but also to minimize potential undesirable outcomes (Gersch, Kreuzer, & Sieber, 2012). To that end, many developments and breakthroughs in target identification techniques have taken place, leading to the elucidation of molecular targets for many drugs and natural products through a variety of approaches (Lomenick, Olsen, & Huang, 2011). One such approach is that of activitybased protein profiling (ABPP), a chemical proteomics approach in which synthetic chemistry is used to generate reactive probes based on the compound of interest which are capable of covalent interaction with their respective molecular targets (B€ ottcher, Pitscheider, Sieber, 2010; B€ ottcher & Sieber, 2010; Krysiak & Breinbauer, 2012; Krysiak et al., 2012; Liu & Guo, 2014; Wirth & Schmuck, 2012). Here, we present a quantitative method whereby probes are functionalized with chemical modifications that allow for bioorthogonal “click chemistry” ligation reactions (exemplified by the copper-catalyzed azide-alkyne cycloaddition reaction or CuAAC), allowing linkage to affinity tags such as biotin or reporter tags such as fluorophores (Rostovtsev, Green, Fokin, & Sharpless, 2002; Sletten & Bertozzi, 2009). This modification enables a two-step process in which protein targets that have been tagged with a probe can either be enriched for target identification or otherwise labeled for visualization (Ovaa et al., 2003; Sletten & Bertozzi, 2009; Zhang, Wang, Ng, Lin, & Shen, 2014). The captured protein targets can then be detected by highsensitivity mass spectrometry (MS) and analyzed with bioinformatics tools and protein databases (Wang, Lee, et al., 2015; Wang, Zhang, Lee, et al., 2016). Compared to classical affinity chromatography methods, this approach allows for analysis of live cells as opposed to cell lysates, ensuring a closer approximation to biological conditions with respect to protein abundance and cellular environment (Wang et al., 2014). The drawback of high background readings from high-throughput MS analysis is offset with the isobaric tag for relative and absolute quantification (iTRAQ) quantitative technique, which can be used to differentiate specific binding targets from nonspecific binding proteins (including endogenously biotinylated proteins) by the use of a control experiment (Wang, Gao, Lee, et al., 2016; Wiese, Reidegeld, Meyer, & Warscheid, 2007). This is a comprehensive and sensitive protocol for target identification of small molecule drugs or natural products in a range of model systems. In this chapter, a detailed protocol for target identification using ABPP and bioorthogonal click chemistry with iTRAQ quantitation will be outlined, based on a previous study which successfully applied the technique

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to elucidate the targets of the bioactive natural compound andrographolide (Wang et al., 2014). In brief, following probe design and synthesis, two replicates of treatment (probe) and control (DMSO) were prepared. Following harvesting and cell lysis, samples were “clicked” with biotin tags which ligate with the modified probes, allowing proteins covalently interacting with the probes to be enriched on avidin beads. Samples were then digested and labeled with different iTRAQ reagents (iTRAQ 113 and 114 for the two controls, and iTRAQ 116 and 117 for the two treatment replicates). Finally, the labeled samples were pooled and subjected to liquid chromatography– tandem mass spectrometry (LC–MS/MS) analysis for protein identification and quantitation. Specific targets (labeled with the 116 and 117 reagents) will exhibit a relatively higher intensity compared to the control (113 and 114), while nonspecific binding proteins will exhibit consistent intensities across treatment and control samples (Fig. 1). While the protocol given is written with reference to the andrographolide study, the steps are generalized and meant to be readily adapted for use with other compounds of interest and in different model cell lines as required (Wang, Zhang, Chia, et al., 2015; Wang, Zhang, Zhang, et al., 2015; Wang, Zhang, Zhang, et al., 2016).

2. MATERIALS, EQUIPMENT, AND SOLUTIONS 2.1 Materials • • • • • • • • • • • •

HCT116 cells are commercially available from ATCC (ATCC® CCL247™, Manassas, VA) Dulbecco’s modified Eagle’s medium: DMEM, containing 4500 mg/L D-glucose (Sigma-Aldrich, St. Louis, #D1152) Heat inactivated fetal bovine serum (FBS; HyClone, South Logan, UT, #SV30160.03) Penicillin–streptomycin mixture (Life Technologies, Carlsbad, CA, #15140122) 1  PBS, pH 7.2–7.4 (Life Technologies, #10010-023) Milli-Q water Molecular biology-grade water (Sigma-Aldrich, #W4502) Dimethyl sulfoxide (DMSO; Sigma-Aldrich, #D2650) Click-iT®AHA (L-azidohomoalanine) reagent (Invitrogen, Carlsbad, CA, #C10289) Trypsin–EDTA, 0.25% (Life Technologies, #25200) Biotin alkyne (Invitrogen, #B10185) Tris (2-carboxyethyl) phosphine hydrochloride, 98% purity (TCEP; Sigma-Aldrich, #C4706)

Fig. 1 General workflow of the quantitative chemical proteomics process with iTRAQ quantitation. Two replicates of treatment (probe) and control (DMSO) are prepared. Following harvesting and lysis, samples are “clicked” with biotin tags, which ligate with the modified probes, allowing proteins covalently interacting with the probes to be enriched on avidin beads. Samples are then digested and labeled with different iTRAQ reagents. Finally, the labeled samples are pooled and subjected to LC–MS/MS analysis for protein identification and quantitation. Specific targets (labeled with the 116 and 117 reagents) will exhibit a relatively higher intensity compared to the control (113 and 114) while nonspecific binding proteins will exhibit consistent intensities across treatment and control samples.

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Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; SigmaAldrich, #678937) Copper sulfate, 99.99% purity (CuSO4; Sigma-Aldrich, #451657) Sodium dodecyl sulfate (SDS; Sigma-Aldrich, #862010) Halt™ Protease Inhibitor Cocktail (Pierce, Waltham, MA, #88266) DNase (New England Biolabs, Ipswich, MA, #M0303S) RNase (Qiagen, Venlo, Netherlands, #19101) Streptavidin beads (Sigma-Aldrich, #S1638) Urea (Sigma-Aldrich, #U5378) Methyl methanethiosulfonate (MMTS; Pierce, #23011) Trypsin, 12.5 ng/μL (Promega, Madison, WI, #V5111) Triethylammonium hydrogen carbonate buffer, 1 M (TEAB; SigmaAldrich, #T7408) iTRAQ Method Development Kit (SCIEX, Foster City, CA, #4352160) Acetonitrile (ACN; Sigma-Aldrich, #34967) Formic acid (FA; Sigma-Aldrich, #V800192) Phosphoric acid (Sigma-Aldrich, #V800287) Acetone (Sigma-Aldrich, #34850) Sodium hydroxide (NaOH; Sigma-Aldrich, #306576)

2.2 Equipment • • • • • • • • • • • •

Biological safety level 2 tissue culture hood (Thermo Fisher Scientific, Waltham, MA) Cell incubator at 37°C, 5% CO2 (Thermo Fisher Scientific) Thermo Scientific Nunc Cell Culture/Petri Dishes 150 mm Dish (Thermo Scientific, Waltham, MA, #1256590) Centrifuge tubes with screw caps, 15 mL (BD Biosciences, Franklin Lakes, NJ, #352196) Refrigerated centrifuge (Eppendorf, Hamburg, Germany, Model 5415R; Model 5810 R) Microcentrifuge tubes, 1.5 mL (Axygen, Corning, NY, #311-08-051) Shaker for microcentrifuge tubes with temperature control (Eppendorf Thermomixer® C from Eppendorf ) Refrigerators, 4°C Freezer, 20°C Vortex mixer (LP Vortex Mixer from Thermo Scientific, #88880018) Minisart filters, 0.45 μm pore size (Sartorius, G€ ottingen, Germany, #51123103) Steritop-GP Filter Unit (Millipore, Billerica, MA, #SCGVT05RE)

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Water bath, 37°C Ultrasonic water bath cleaner (Ultrasonic Cleaner Elmasonic, Maplewood, NJ, SH250EL) Phase-contrast microscope (Nikon ECLIPSE, Tokyo, Japan, TE2000-S) MicroSpin Columns (GEHealthcare, Chicago, IL, 27-3565-01) TripleTOF 5600 system (AB SCIEX, Foster City, CA) Parafilm C18 Sep-Pak column (Waters, Milford, MA, #WAT051910) ChiPLC-nanoflex (Eksigent, Dublin, CA) Speedvac (Savant™ SPD131DDA SpeedVac™ Concentrator from Thermo Scientific, #SPD131DDA-115) Lyophilizer machine (American Lyophilizer, Inc., Yardley, PA) Eksigent nano-liquid chromatography (nano-LC)-Ultra system coupled to a the ChiPLC-nanoflex system (Eksigent) ProteinPilot™ 4.5 (AB SCIEX) Probe sonicator (Hielscher–Ultrasound Technology, Teltow, Germany) Chemical fume hood (Flow Sciences, Inc., Leland, NC) Cation Exchange Buffer Pack (AB SCIEX, #4326747), containing individual 100 mL bottles of loading buffer, elution buffer, cleaning buffer, and storage buffer. The Buffer Pack also includes one 0.2 mL cation exchange cartridge. Cation exchange column (AB SCIEX, #4326695)

2.3 C18 Buffers (for Section 3.6) C18 buffer A: 98% H2O, 2% ACN, 0.05% FA. Elution buffer E1: 50% ACN, 50% H2O; Elution buffer E2: 75% ACN, 25% H2O.

2.4 Gradient Separation Buffer (for Section 3.7) Mobile phase A: 2% ACN, 0.1% FA. Mobile phase B: 98% ACN, 2% H2O, 0.05% FA.

3. PROTOCOL 3.1 Cell Culture and Probe Treatment 1. For this protocol, culture HCT116 human colon carcinoma cells in DMEM (10% FBS, 5% CO2 incubation at 37°C) to 80% confluence in 150 mm dishes.

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2. Discard culture media and wash cells with PBS. Perform the necessary probe treatments under appropriate conditions with reference to the original drug. 3. Use DMSO as a control and prepare at least two treatment and two control samples (see Note 1). 4. Following treatment, wash cells with PBS and trypsinize to detach cells from the culture dishes. Centrifuge at 1200  g to pellet the cells and discard the supernatant. 5. Lyse the cells with 2 mL 0.16% SDS in PBS with 50 μg/mL DNase and 50 μg/mL RNase, 1  Halt™ Protease Inhibitor Cocktail. Sonicate the cell suspension with 1-s pulses for 60 s (see Note 2). 6. Remove the insoluble fraction following centrifugation at 10,000 rpm for 45 min. Retain the protein-containing supernatant, which can be stored at 80°C if necessary. Note 1: Other controls using original drug or the inactivated drug analogue probes can also be used. Multiple treatment and control samples serve as biological replicates to account for experimental variations and improve the reliability of the target identification. Note 2: If SDS is used for the cell lysis, ensure that it is diluted to a final concentration of 0.2% or below as it can interfere with subsequent click chemistry reactions.

3.2 Click Chemistry Tagging With Biotin Alkyne 7. Perform protein quantitation (e.g., Bradford method) for each sample from step 6. Use an equal amount of protein (4 mg) for each sample to perform the subsequent click chemistry tagging steps. 8. Top up each sample to a volume of 2 mL with 1  PBS. 9. Prepare the following chemicals required for the click reaction: 100  biotin alkyne (10 mM in DMSO), 100  TCEP (100 mM in water), 100  TBTA (10 mM in DMSO), and 100  CuSO4 (100 mM in water). See Note 3 for additional information on preparation. 10. Add 20 μL of 100  biotin alkyne to each sample and vortex. 11. Add 20 μL of 100  TCEP in water (from step 9) and vortex. 12. Add 20 μL of 100  TBTA in DMSO (from step 9) and vortex. 13. Add 20 μL of 100  CuSO4 (from step 9) and vortex. 14. Incubate the prepared samples for 2 h at room temperature, under dark conditions with constant gentle mixing. 15. For protein precipitation, transfer each sample to a 15-mL centrifuge tube and top up with 10 mL of prechilled (20°C) acetone.

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16. Incubate the samples at 20°C for 4 h. 17. Centrifuge for 30 min at 4000  g at 4°C. 18. Carefully decant the supernatant without dislodging the protein pellet. Allow the remaining acetone to air-dry at room temperature (see Note 5). 19. Dissolve the pellets in 700 μL of 1.2% SDS in PBS with sonication, at 0.5-s pulses for 60 s. Heat the samples for 5 min at 80–90°C (see Note 6). 20. Dilute the dissolved samples in 10 mL PBS (see Note 7). 21. Centrifuge the samples in room temperature at 4000  g. Collect the supernatant for the subsequent steps (see Note 8). Note 3: Use freshly prepared CuSO4 and TCEP. Biotin alkyne (10 mM) and TBTA (10 mM) stocks can be prepared in advance and stored in 20°C up to 2 months for future use. Note 4: In addition to precipitating the protein, the acetone precipitation step is important for removing free biotin tags from the mixture. Free biotin tags can interfere with avidin binding in the subsequent affinity enrichment steps and significantly decrease the efficiency of enrichment. Note 5: Avoid overdrying the samples as that will make the following redissolving step more difficult. Note 6: The high SDS concentration and heating steps may be necessary to ensure complete protein dissolution. Note 7: As mentioned previously, it is important to keep the SDS concentration below 0.2% to minimize effects on subsequent click chemistry procedures. Note 8: Samples can be stored for several days if necessary at 80°C.

3.3 Streptavidin Affinity Purification 22. For each sample, prepare and wash 70 μL of Streptavidin beads. Add 70 μL of the Streptavidin beads to a 15-mL centrifuge tube and wash with 5 mL PBS. Centrifuge at 700  g for 3 min at room temperature after washing (see Note 9). 23. Perform two more repetitions of the wash step. 24. Add the protein sample obtained from step 21 to the washed beads and incubate at room temperature with gentle rotation using a rotator for 4 h. 25. Centrifuge for 3 min at 700  g for 3 min. Carefully discard the supernatant without removing the beads.

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26. Wash the beads with 10 mL of 1% SDS in PBS. Gently mix on a rotator or shaker for 10 min, then centrifuge at 700  g for 5 min at room temperature to pellet the beads. Carefully discard the supernatant. 27. Perform two more repetitions of the previous wash step (step 26). 28. Wash the beads with 10 mL of 6 M urea. Gently mix on a rotator or shaker for 10 min, then centrifuge at 700  g for 5 min at room temperature to pellet the beads. Carefully discard the supernatant. 29. Perform two more repetitions of the previous wash step (step 28). 30. Wash the beads with 10 mL PBS. Gently mix on a rotator or shaker for 10 min, then centrifuge at 700  g for 5 min at room temperature to pellet the beads. Carefully discard the supernatant. 31. Perform two more repetitions of the previous wash step (step 30). 32. Finally, wash the beads with 10 mL Milli-Q water. Gently mix on a rotator or shaker for 10 min, then centrifuge at 700  g for 5 min at room temperature to pellet the beads. Carefully discard the supernatant. 33. Reconstitute the beads in 200 μL of 0.5 M TEAB. 34. Reduce the mixture by adding 4 μL TCEP to each sample. Vortex the mixtures and incubate at 60°C on a Thermo shaker at 800 rpm for 60 min. Allow the samples to cool at room temperature. 35. Add 2 μL MMTS (200 mM) to each sample for cysteine blocking. Vortex the mixtures and incubate at room temperature for 15 min (see Note 10). 36. Prepare 0.5 μg/μL trypsin in water (see Note 11). 37. Add 8 μL (4 μg) of the trypsin solution into each sample. Incubate at 37°C for 16 h to digest the proteins captured on the beads (see Notes 12 and 13). 38. Isolate the digested peptides from the mixture using a filter spin column. Collect the sample solution containing the digested peptides. 39. The samples can now be used for the subsequent iTRAQ labeling step, or otherwise be stored at 80°C for future use. Note 9: It is important to maintain a centrifugation speed of no more than 1000  g for all subsequent wash steps involving the streptavidin beads as the beads are fragile and can be damaged. Note 10: MMTS is toxic and should be handled in an appropriate chemical hood. Note 11: Use sequencing grade modified trypsin. Note 12: Seal the sample tubes with parafilm to prevent sample loss through evaporation. Note 13: Ensure a minimum final trypsin concentration of 10 ng/μL for each sample.

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3.4 (iTRAQ) Labeling 40. Evaporate the samples obtained from step 39 using a vacuum concentrator (SpeedVac™) device. Several hours may be needed to fully dry the samples (see Notes 14 and 15). 41. Reconstitute the dried samples in 30 μL TEAB (250 mM). Vortex and spin the samples, ensuring the solution is clear (see Note 16). 42. Adjust the pH of the samples to 8 (alkaline) to ensure optimal labeling (see Note 17). 43. Prepare the iTRAQ reagents (4- or 8-plex) according to manufacturer’s instructions. Allow reagents to warm to room temperature and reconstitute in 70 μL ethanol (4-plex) or 50 μL isopropanol (8-plex). 44. Perform the iTRAQ labeling as per manufacturer’s instructions. Use a 1-h reaction time for the 4-plex kit and 2 h for the 8-plex kit (see Notes 18 and 19). For this protocol, the labeling will be referenced as follows: iTRAQ 113 for control 1, iTRAQ 114 for control 2, iTRAQ 116 for treatment 1, and iTRAQ 117 for treatment 2. 45. Pool all iTRAQ-labeled samples together in a single new tube. Note 14: It is important to standardize the sample volumes through this step for the subsequent labeling procedures. Note 15: The dried peptide sample can be stored for several months at 80°C. Note 16: Sonicate sample to dissolve any precipitates. Note 17: pH indicator strips are sufficient for pH reading. Use NaOH to adjust the pH to 8 if necessary. Note 18: Take great care to ensure complete transfer of the iTRAQ reagent to the sample, maintaining equal volume across samples. Note 19: Avoid overlabeling. Add phosphoric acid to quench the reaction after the necessary duration.

3.5 Sample Clean up by Strong Cation Exchange Chromatography 46. Centrifuge the pooled sample from step 45 at 14,000  g for 10 min. Transfer the supernatant to a 50-mL Falcon tube and dilute the sample by approximately 10 times in Strong Cation Exchange (SCX) Load Buffer (see Notes 20 and 21). 47. Check and adjust the pH of the sample to between 2.5 and 3.3 (see Note 22). 48. Clean and condition the SCX cartridge by injecting 1 mL buffer-clean into the cartridge. Divert the flow-through to waste.

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49. Inject 2 mL Load buffer and divert flow-through to waste. 50. Slowly (1 droplet per second) inject the diluted sample from step 46 (ensuring there are no air bubbles) into the cartridge. Collect flowthrough in a fresh 50 mL-tube. 51. Inject 1 mL Load buffer and collect flow-through in the 50-mL tube. This is to remove excess reagents such as TCEP, SDS, and iTRAQ reagents from the SCX cartridge (see Note 23). 52. Slowly inject 500 μL Elute buffer and capture the eluate in a fresh 1.5 mL-sample tube. 53. Inject 1 mL cleaning buffer to wash the cartridge. Divert flow-through to waste. 54. Inject 2 mL storage buffer and divert flow-through to waste. Disassemble and store the apparatus at 4°C. Note 20: Avoid injecting any precipitates into the column, which can cause column clogging and sample loss. Note 21: All SCX buffers should be stored at 4°C. Note 22: Handle phosphoric acid in a fume hood. Note 23: Retain this flow-through until it can be verified by MS/MS analysis that the sample loading onto the SCX cartridge was successful. This can be used to repeat the loading as necessary.

3.6 Desalting of Labeled Samples by C18 Column 55. Dilute the sample collected from step 52 in a 15-mL centrifuge tube with 3–4 mL C18 buffer A (from 2.5). Ensure that all sample is transferred by rinsing the original 1.5-mL tube a few times. 56. Rinse a fresh 5-mL syringe with 100% ACN. Connect the syringe to the short end of a Sep-Pak C18 cartridge column. Condition the column with 100% ACN (10 mL) (see Note 24). 57. Condition the Sep-Pak column by injecting 10 mL of C18 buffer A (from 2.5) into the column. 58. Slowly inject (1 drop per second) the diluted sample into the column and collect the flow-through. Repeat the process once, loading the flow-through into the column and collecting this second flowthrough. 59. Inject 5 mL C18 buffer A (from 2.5) into the column in order to remove excess salts and other contaminants. 60. Inject 5 mL elution buffer E1 and 5 mL elution buffer E2 (from 2.5). Collect the flow-through in a fresh 50-mL Falcon tube.

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61. Add equal volume (10 mL) Milli-Q water to the flow-through from the previous step. Gently swirl the tube and freeze overnight at 80°C. 62. Remove the cap of the sample tube. Cover the tube tightly with parafilm, wrapping several times across the opening. Lightly perforate the parafilm with a needle (about 5–10 holes). 63. Leave the sample to lyophilize overnight in a lyophilizer. 64. Reconstitute the lyophilized sample in 1.5 mL of mobile phase A (2% ACN, 98% water). 65. Transfer the sample to a 2-mL Eppendorf tube. Dry the sample with a SpeedVac concentrator (see Note 25). Note 24: Ensure at all points during this procedure to avoid introducing air bubbles into the column, which will significantly affect its binding capacity. Note 25: The dried sample can be stored for several months if necessary at 80°C.

3.7 Nano-LC Electrospray Ionization MS 66. Reconstitute the dried sample from step 65 in 100 μL of C18 buffer A (from 2.5). 67. Perform the nano-LC separation of iTRAQ-labeled peptides. Suggested equipment: Eksigent NanoLC-Ultra system coupled to the cHiPLC-Nanoflex system (Eksigent), with a 75 μm  150 mm analytical column packed with ReproSil-Pur C-18 (Eksigent, 804–00011) set to the “Trap-Elute” configuration (see Note 26). Load 5 μL of the reconstituted sample in the LC system. 68. Prepare mobile phase B (98% ACN, 0.1% formic acid) for the gradient elution step. Use the following parameters for the peptide separation: 5–12% mobile phase B for 20 min, 12–30% mobile phase B for 90 min, and 30–90% mobile phase B for 2 min, followed by regeneration of the column at 90% mobile phase B for 5 min, 90–5% mobile phase B for 3 min, and finally 5% mobile phase B for 13 min. Set flow rate to 300 nL/min. 69. Acquire MS and MS/MS spectra with a suitable apparatus such as the TripleTOF 5600 (SCIEX) (see Note 27). The parameters are described as follows: one 250 ms MS analysis followed by MS/MS analyses of 20 precursors with 100 ms accumulation time per spectrum, for each duty cycle. For MS spectra, use high-resolution mode with the mass range set at 350–1250 m/z. For MS/MS spectra, use high-sensitivity

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mode with a mass range of 100–1800 m/z and a dynamic exclusion of 15 s. Select rolling collision energy and iTRAQ reagent collision adjustment settings for MS/MS analyses (Wang, Lee, et al., 2015; Wang, Zhang, Chia, et al., 2015; Wang, Zhang, Zhang, et al., 2015). Note 26: Other nano-LC equipment can also be used as necessary. Note 27: The SCIEX TripleTOF 5600 and 6600 series mass spectrometers are optimized for iTRAQ analysis. If other models are used, ensure that they are compatible with iTRAQ analysis and have the ability to detect iTRAQ reporter ions at m/z 113–121 with good resolution, as well as suitable collision energy to fragment iTRAQ-labeled peptides and produce the necessary reporter ions).

3.8 Protein Identification and Quantification Using ProteinPilot™ Software 70. Perform peptide identification and quantification using the ProteinPilot™ Software 4.5 with the Paragon algorithm (4.5.0.0, 1654) (see Note 28). Use the SwissProt (v2015.9) database with the following parameters enabled: Cysteine alkylation with MMTS; trypsin digestion; TripleTOF 5600; biological modifications (see Notes 29 and 30). 71. Export the protein summary file into Microsoft Excel or any other suitable tool for data analysis. 72. Filter the protein list with a total unused score cutoff of 1.3, corresponding to 95% confidence level. 73. Derive the treatment vs control iTRAQ ratios for all samples. For the labeling described in step 44, the ratios will thus be (116:113, 116:114, 117:113, and 117:114). iTRAQ ratios are then converted to log2 figures for statistical analysis. 74. Perform a 1-sample t-test to check for the significance of the data (see Note 31). 75. Use p  0.05 as the significance cutoff. 76. Eliminate proteins with a 114/113 or 117/116 (that is, between control and between treatment samples) ratio of 1.3 or 0.77. These results are inconsistent between replicates and considered unreliable (see Note 32). 77. Eliminate proteins identified by a single unique peptide which may be unreliable (see Note 33).

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78. Eliminate proteins with a mean iTRAQ ratio (treatment over control) <2 (see Note 34). The remaining proteins at this point are positive results. Note 28: The ProteinPilot Software is recommended for analysis of iTRAQ data. In the event that other software needs to be used, ensure that only unique peptides are used to calculate the iTRAQ ratios. Exclude peptides with incomplete iTRAQ labeling, trypsin miscleavage or methionine oxidation. Note 29: The false discovery rate of the peptide identification can be estimated with a decoy database search. The ProteinPilot Software 4.5 is able to do this through its Proteomics System Performance Evaluation Pipeline feature. Note 30: Do not normalize the iTRAQ ratio. If ProteinPilot is used, background correction is not recommended. Note 31: In Excel, for each set of four log2 ratios associated with each protein, create four 0 values and then perform two-tailed paired t-test. This is equivalent to 1-sample t-test. Note 32: These cut-off thresholds are derived from a standard deviation of 0.15 for the labeled peptide ratios, using a 1  2 standard deviation formula (Higuchi et al., 2013; Tan et al., 2008; Wang et al., 2014; Wang, Zhang, Zhang, et al., 2016). iTRAQ ratios between replicates (that is, treatment 1 over treatment 2 and control 1 over control 2) that exceed these cutoffs are thus considered inconsistent and eliminated. Note 33: This is a guideline to improve the reliability and robustness of the data. Proteins identified with a single unique peptide can still be positive data and might not necessarily be eliminated. Further experimental support and justification may, however, be necessary for such cases. Note 34: This is the cut-off threshold to determine specific from nonspecific targets, where an enrichment ratio of two or higher suggests that the target is significantly enriched in treatment samples compared to the control. The threshold figure of two is an arbitrarily defined guideline that can be considered highly stringent given the commonly observed ratio compression in iTRAQ experiments.

4. DISCUSSION We have presented a protocol detailing a quantitative chemical proteomics approach to target identification of drugs and natural products, using

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click chemistry-based target pull-down and iTRAQ quantitation (Ross et al., 2004). The principles involved (described briefly in Section 1) have been rapidly refined and successfully applied in the recent years to target identification in a range of drugs and models. The approach taken in this protocol offers several advantages. The use of probes with small click chemistry-enabling modifications rather than bulkier modifications such as biotin tags allows for treatment in live cell conditions instead of cell lysates (Kalesh, Clulow, & Tate, 2015). MS detection also offers a significantly improved resolution and sensitivity over traditional gel-based detection methods (Rix & Superti-Furga, 2009). Finally, iTRAQ quantitation allows for parallel analysis of up to eight samples, allowing the use of biological replicates and control experiments to improve the robustness of the obtained data. At the same time, it is important to note that there exist several other quantitative proteomic approaches that have been successful in their application, including SILAC (stable isotope labeling by amino acids in cell culture), Tandem Mass Tag™ (TMT), SWATH, and other emerging label-free methods which have been covered in several comprehensive reviews (Gillet et al., 2012; Ong et al., 2002; Rix & Superti-Furga, 2009; Wang, Gao, Lee, et al., 2016; Wright & Sieber, 2016). It will be useful to consider these multiple available quantitative proteomics approaches and their respective capabilities for different needs. The method provided here can be readily adapted and modified for use in different model cell lines, as well as different drugs and natural products as long as the appropriate probes can be designed to allow for affinity pull-down without interfering with function. To that end, it is important to understand the structural and chemical properties of the drug in question and identify reactive sites to facilitate probe design. A relevant advantage of click chemistry-enabled probes is that the modifications can be multipurpose, allowing for easy analysis by gel-based fluorescence assays via the attachment of reporters (rather than affinity tags for pull-down) (Liu et al., 2009; Shi, Cheng, Sze, & Yao, 2011, Shi, Zhang, Chen, & Yao, 2012). This allows for probes to be readily tested against the original compound for binding effectiveness and binding specificity, as probes must be shown to mimic the original compound in bioactivity as closely as possible. The versatility of the presented method extends to noncovalent interacting partners—weakly interacting targets can be pulled down by using milder wash conditions (PBS or with low concentration detergent only), expanding the range of target identification at the risk of increased background which may necessitate changes to data analysis.

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ACKNOWLEDGMENTS We acknowledge the Chinese National Natural Sciences Foundation (81421091) and the Doctoral Station Science Foundation from the Chinese Ministry of Education of China (20130091130003). This work was supported in part by the National Research Foundation-supported Interdisciplinary Research group in Infectious Diseases of SMART (Singapore-MIT Alliance for Research and Technology) and by research grants from the National Medical Research Council Singapore (NMRC-CIRG/1346/2012 and NMRC/CIRG/1373/2013) to H.-M.S. Y.-M.L. is supported by NUS Research Scholarships.

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