Supporting InformationOng et al. 10.1073/pnas.0900191106SI MethodsMaterials and Reagents. L-arginine-13C6 and L-lysine-13C615N2were from Sigma Isotec. The cell culture media, Roswell ParkMemorial Institute-1640 (RPMI) deficient in arginine, lysineand methioine, was a custom media preparation from CaissonLaboratories. All other L-amino acids were obtained fromSigma. Dialyzed serum was obtained from SAFC-Sigma. Trypsinwas from Promega and HeLaS3 and H1299 were a kind gift fromDr. James Bradner. A PC-12 rat pheochromocytoma cell linestably expressing hErbB4 and GFP was generated with neomycinselection and 2 independent pcDNA3-neomycin (Invitrogen)constructs. Other cell lines were from ATCC.

Antibodies against the following proteins were used: Anti-FKBP1A and anti-MTAP (Abcam); anti-FKBP2 and anti-FKBP5 R&D Systems); anti-FKBP4 (Bethyl Laboratories); anti-FKBP8 (US Biological); anti-FKBP10 (listed in the article asFKBP9/10) (BD Transduction Laboratories). Secondary anti-bodies were all purchased from Sigma. Anti-GST antibody wasfrom GE Healthcare (Piscataway, NJ). All other reagents andchemicals used were of the highest grade available.

SILAC Media Preparation and Cell Culture Conditions. We followedall standard SILAC media preparation and labeling steps asdescribed in ref. 1. Briefly, 15 mg/L of L-methionine was addedto base media according to standard formulations for standardRPMI. This base media was divided into 2 and either ‘‘light’’forms of arginine and lysine or ‘‘heavy’’ L-arginine-U-13C6 (87.2mg/L) and L-lysine-13C615N2 (155 mg/L) was added to generatethe 2 SILAC labeling mediums. Each medium with the fullcomplement of amino acids was sterile filtered through a 0.22�M filter (Milipore).

HeLa S3 cells were grown in RPMI labeling media, preparedas described above, supplemented with 2 mM L-glutamine, and5% dialyzed FBS plus antibiotics, in a humidified atmospherewith 5% CO2 in air. Cells were grown for at least 6 cell divisionsin labeling media, initially growing in flasks but expanded into2L spinner flasks on a magnetic stir plate to provide largercultures.

Biochemical Purification with Small Molecule Affinity Matrices. Sep-arate cultures of HeLaS3 cells SILAC labeled either withL-arginine and L-lysine (light) or L-arginine-13C6 and L-lysine-13C6-15N2 (heavy) are lysed in ice-chilled ModRIPA buffer (lowstringency buffer LS) containing 1% Nonidet P-40, 0.1% Nadeoxycholate, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5,and protease inhibitors (Complete tablets, Roche Applied Sci-ence, Indianapolis, IN). Lysates are vortexed intermittently whilechilled on ice for 10 min and clarified by spinning at 14,000 � g.Protein concentrations of light and heavy lysates are estimatedwith the Protein Assay Dye Reagent Concentrate (Bio-Rad,Hercules CA) and equalized. The protein concentrations oflysates can vary between 1.7 to 2.2 mg/mL, affinity enrichmentsare performed in lysate volumes between 1 mL to 1.4 mL in a 1.5mL microcentrifuge tube.

In bead control (BC) experiments, 2 mg of light HeLaS3 lysateis incubated with 25 �L of 50% slurry of EtOH-bead while 2 mgof heavy lysate is incubated with 25 �L of 50% small moleculeaffinity beads (SM-bead). This is termed a ‘‘forward’’ labelexperiment. In the ‘‘reverse’’ experiment, the lysates areswapped for each bead type. In a ‘‘forward’’ soluble competitorexperiment, the appropriate amount of small molecule (inDMSO) is added to 2 mg of light HeLaS3 lysate. An equal

volume of DMSO is then added to 2 mg of heavy HeLaS3 as acontrol. 25 �L of 50% of SM-bead is added to both light andheavy lysates in SC experiments.

Affinity enrichments are incubated overnight (�16 h) on anend-over-end rotator at 4 °C. After incubation, the tubes arespun at 1000 � g on a benchtop centrifuge to pellet the beads.The supernatant is aspirated, taking care to avoid disturbing thebeads. In BC experiments, beads are combined at the first washfor subsequent washing steps. For SC experiments, each tube ina set is washed with ModRIPA buffer at least once (twice withhigh levels of soluble competition) to remove excess solublesmall molecule competitor. Beads from the 2 tubes were then becombined for later washing steps. In high stringency experi-ments, a wash buffer (high stringency HS) containing ModRIPAand 0.2% SDS was used to wash beads instead. After the thirdand final wash, beads are collected by spinning at 1000 � g andthe wash is aspirated leaving �20 �L of buffer in the tube.

1D-SDS/PAGE and MS Analysis. Proteins enriched in SILAC affinitypull-downs were reduced and alkylated, on bead, in 2 mM DTTand 10 mM iodoacetamide respectively. One part LDS buffer(Invitrogen) was added to 3 parts sample (including beads) andtubes heated to 70 °C for 10 min. Proteins were resolved on a4–12% gradient 1.5 mm thick Bis-Tris gel with Mes runningbuffer (Nupage, Invitrogen) and Coomassie stained (SimplyBlue, Invitrogen). Gel lanes were excised into 6 pieces and thenfurther cut into 1.5 mm cubes. The gel pieces were furtherdestained in a solution containing 50% EtOH and 50% 50 mMammonium bicarbonate, then dehydrated in 100% EtOH beforeaddition of sufficient trypsin (12.5 ng/�L) to swell the gel piecescompletely. An additional 100 �L of 50 mM ammonium bicar-bonate was added before incubating at 37 °C overnight on athermomixer (Eppendorf). Enzymatic digestion was stopped bythe addition of 100 �L of 1% TFA to tubes. A second extractionwith 300 �L of 0.1% TFA was combined with the first extract andthe peptides from each gel slice cleaned up on C18 StageTips (2).Peptides were eluted in 50 �L of 80% acetonitrile/0.1% TFA anddried down in a evaporative centrifuge to remove organicsolvents. The peptides were then resuspended by vortexing in 7�L of 0.1% TFA and analyzed by nanoflow-LCMS with anAgilent 1100 with autosampler and a LTQ-Orbitrap. Peptideswere resolved on a 10 cm column, made in-house by packing aself-pulled 75 �m I.D. capillary, 15 �m tip (P-2000 laser basedpuller, Sutter Instruments) column with 3 �m Reprosil-C18-AQbeads (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)with an analytical f lowrate of 200 nL/min and a 58 min lineargradient (�0.57%B/min) from 0.1% formic acid in water to 0.1%formic acid/90% acetonitrile. The run time was 108 min for asingle sample, including sample loading and column recondi-tioning.

We used a MS method with a master Orbitrap full scan (60,000resolution) and data dependent LTQ MS/MS scans for the top5 precursors (excluding z � 1) from the Orbitrap scan. Each cyclewas �2 secs long. MS raw files were processed for proteinidentification and quantitation using extract�msn.exe (Thermo),Mascot (Ver. 2.1.03 Matrixscience), and academic software,DTASupercharge and MSQuant (CEBI, open-source http://msquant.sourceforge.net). MS/MS peak lists in Mascot GenericFormat were generated using extract�msn.exe and DTASuper-charge and searched with Mascot using IPI human ver.3.32(http://ebi.ac.uk) with a concatenated decoy database containingrandomized sequences from the same database. Common con-

Ong et al. www.pnas.org/cgi/content/short/0900191106 1 of 15

taminants like BSA, trypsin etc. were also added to the database.Variable modifications used were oxidized methionine, argi-nine-13C6, lysine-13C6

15N2, and carbamidomethyl-cysteine was afixed modification. The precursor mass tolerance used in thesearch was 15 ppm and fragment mass tolerance was 0.7 Da.Proteins with a minimum Mascot score of 66 (at least 1 peptidewith score �66) and peptides with score �20 were quantified byMSQuant. Only proteins with a minimum of 2 quantifiablepeptides were included in our dataset. The false positive rate forprotein identification is �1% and �5% at the peptide level, asdetermined using the decoy database strategy.

Statistical Analysis of SC Experiments. To model log2 protein ratiovalues from SC experiments, we adapted the empirical Bayesframework developed by Efron (3) to compute the posteriorprobability that a ratio value arises from the null distribution.Briefly, by application of Bayes’s theorem, this quantity iscomputed as Pr(Z � 0�X) � [Pr(X�Z � 0)Pr(Z � 0)]/Pr(X) whereZ is a binary variable taking the value of 1 if the protein is boundby the compound, and X is a measured log2 SILAC ratio. Wemodel Pr(X) using a Gaussian kernel estimator with paretodistributions fit to 5% of the data at either tail, to avoidunreliable estimation in regions of data sparsity. The distributionfor Pr(X�Z � 0)Pr(Z � 0) is then inferred by fitting a Gaussiandistribution using only the portion of Pr(X) arising from thecentral two thirds of the data.

Annotation of Kinases as ‘‘True Positive’’ Set. We use the humankinome defined in ref. (4), specifically, the updated Excelspreadsheet (http://kinase.com/human/kinome/tables/Kincat�Hsap.08.02.xls). We note that some of our identifiedtargets were not on this list because they were regulatorysubunits of a kinase (for e.g., PRKAR1A, PRKAR2A andPHKB).

Western Blot Analysis Experiments. Western blot analysis experi-ments to validate affinity enrichment were performed in thesame manner as with SILAC experiments, except that cellsgrown with normal RPMI-1640 media (Invitrogen) was used.The cells were grown, lysed and-treated identically to the SILACTargetID experiments as described above. After separation ofSM-bead enriched protein on SDS/PAGE, proteins were trans-ferred to a nitrocellulose membrane using a semidry blottingapparatus (Bio-Rad). The membrane was blocked with 5% skimmilk powder in PBS-Tween, washed in 0.1% PBS-T, and thenincubated with the corresponding antibodies, as indicated inFigs. 5 and 6 and Fig. S5, followed by HRP-conjugated secondaryantibodies. The membranes were subjected to chemiluminescentdetection according to manufacturer’s instructions (ECL, GEHealthcare).

MTAP Functional Assay. We used a published method (5) formeasuring MTAP activity with slight modifications. Briefly,MTAP’s production of adenine from MTA is monitored after itsconversion to 8-dihydroxyadenine by xanthine oxidase. Wemeasured change in absorbance at 305 nm in a 96-well plateusing a SpectraMax Plus (Molecular Devices, Sunnyvale, CA).For soluble competition, either a DMSO control, 10� (225nmoles), 50� (1125 nmoles), or 100� (2250 nmoles) of each ofthe IPL ligands was added to �2 mg or 1 mL of cell lysate andincubated for 1 hour on an end-over-end rotator at 4 °C. Assayswere done at 37 °C in a total volume of 300 �L containing 100�L of cell lysate (containing small molecule or control), 24 �Lor 0.8 units of xanthine oxidase (Sigma), 176 �L of 40 mMKH2PO4 and10 �M MTA. Kinetic Vmax plots were acquired overa 25 min acquisition period and plotted using SpectraMaxsoftware.

FKBP10/FKBP9 Antibody Cross-Specificity Determined by Immunopre-cipitation and MS (IP-MS). In Western blot analysis experiments tovalidate our IPL ligand SILAC TargetID data, we found theFKBP10 antibody (BD Transduction Laboratories, Cat. No.610648) yielded a strong band at �63 kDa in IPL ligandpull-downs with AP-1480, ProAP-1480, and AP-1497. This wasnot consistent with our SILAC data for FKBP10, but matchedthe compound specificity profile for FKBP9. Because FKBP9and FKBP10 are very similar in length, 570 aa and 582 aarespectively and are 58% identical at the amino acid level(BlastP), we hypothesized that the antibody used in our exper-iment was cross-reacting with both proteins. We evaluated thiswith an IP-MS experiment. We incubated 10 �g of antibody and50 �L of a 50% slurry of Protein G beads with 2 mg of HeLaS3lysate in our usual pull-down conditions. We used ProteinG-bead alone with lysate in a separate pull-down as a control.Beads were washed, proteins reduced, alkylated, resolved on a1D-SDS/PAGE gel and the molecular mass bands between �60to 98 kDa were excised for MS analysis. Our MS analysisidentified both FKBP9 and FKBP10 in the pull-down with theantibody and strikingly, the number of peptides and sequencecoverage for FKBP9 was higher than FKBP10.

Surface Plasmon Resonance Experiments. The surface plasmonresonance assays were conducted on a Biacore S51 instrumentusing Biacore CM5 sensor chips. Ethanolamine, EDC, NHS, andP-20 surfactant were all obtained from GE Lifesciences. Anti-GST antibody (GE LifeSciences) was directly immobilizedthrough primary amines using standard EDC/NHS chemistryaccording to the manufacturer’s instructions. Either MTAP-GST (BPS Biosciences) or GST was captured to generate theMTAP or reference surfaces. Sensor data were analyzed usingScrubber 2 software (BioLogic Software Pty Ltd.) or BiaEvalu-ation (GE LifeSciences). Data were double-reference subtractedand corrected for variation in solvent concentration. Bindingaffinity was calculated using kinetic and equilibrium analyses.Kinetic analysis was performed using a least-squares fit of aLangmuir 1:1 binding model.

Sensor Chip Preparation. The sensor surface was conditioned usingalternating injections of 10 mM glycine pH 2.2 and 50 mmNaOH. The surface was then activated with 1:1 4 M EDC/1 MNHS for 10 min. Anti-GST antibody was diluted to 30 �g/mL in10 mM acetate pH 5.0 and was exposed to the activated surfacefor 10 min at 5 �L/min. The surface was quenched by a 7 mininjection of 1M ethanolamine. Between 13,000 and 14,500response units (RU) were immobilized for each assay. MTAP-GST and GST were diluted to 75 �g/mL and 7.5 �g/mL,respectively and captured on the anti-GST antibody surface witha 10 min injection at 5 �L/min. Between 1,500 and 1,700 RU ofprotein were captured for each assay. The running buffer usedduring immobilization and capture was PBS, pH 7.4 with 0.005%P-20 surfactant.

SPR Binding Assay Parameters. Small-molecule binding assays wereperformed at 25 °C. The running buffer for the binding assayswas PBS, pH 7.4 with 0.005% P-20 surfactant and 2% DMSO asa cosolvent. Compounds were diluted from 10 mM stocks inDMSO to the appropriate concentration in buffer with the samesolvent concentration as the running buffer (2%). Binding wasmeasured at concentrations from 4.8 nM to 20 �M in halfdilutions. Compound was injected for 120 sec followed by 120 secof buffer with no compound.

Preparation of Bait Compounds and General Procedure of Solid-PhaseImmobilization. All chemicals were purchased from Sigma–Aldrich and common solvents were purchased from Fisher(J.T.Baker) at HPLC grade unless otherwise noted. Automated

Ong et al. www.pnas.org/cgi/content/short/0900191106 2 of 15

f lash chromatography was performed on Teledyne ISCO Com-biFlash Rf systems. HRMS data were collected on a BrukerDaltonics APEXIV 4.7 FT-ICR mass spectrometer. 1H and13C-NMR spectra were collected on a Bruker 300 MHz NMRspectrometer.

(S)-((R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(3-hydroxypropylamino)-2-oxoethoxy)phenyl)propyl) 1-(3,3-dimethyl-2-oxopentanoyl)piperi-dine-2-carboxylate, AP-1497 alcohol (Fig. S1B). Starting material,2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-((S)-1-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carbonyloxy)propyl)phenoxy)aceticacid, was prepared with reported synthetic procedure (6).

The starting acid (103 mg, 0.176 mmol, 1 eq.) and CDI (34.2mg , 0.211 mmol, 1.2 eq.) were dissolved in anhydrous DMF (2mL) under nitrogen. The mixture was stirred at room temper-ature for 15 min to activate the acid. 3-Amino-1-propanol (67.3�L, 66.1 mg, 0.880 mmol, 5.0 eq.) was added to the solution andthe reaction was maintained at room temperature for 1 h. Thereaction mixture was then concentrated and purified by auto-mated flash chromatography to afford the pure product (93.2mg, 83%). 1H NMR (300 MHz, CDCl3) � 7.30 (m, 2H), 6.94 (t,2H, J � 9.7), 6.80 (m, 2H), 6.68 (dd, 2H, J � 3.4, 7.4), 5.76 (dd,1H, J � 5.5, 7.9), 5.30 (d, 1H, J � 4.9), 4.45 (d, 2H, J � 28.5),3.81 (dd, 6H, J � 3.5, 19.5), 3.65 (t, 2H, J � 5.3), 3.50 (dd, 2H,J � 6.1, 12.1), 3.35 (s, 2H), 3.17 (dd, 1H, J � 7.9, 17.9), 2.94 (s,1H), 2.86 (s, 1H), 2.57 (m, 2H), 2.36 (d, 1H, J � 13.5), 2.23 (dt,1H, J � 8.8, 14.3), 2.07 (m, 2H), 1.69 (m, 7H), 1.42 (m, 2H), 1.21(t, 5H, J � 3.6), 1.14 (s, 1H), 0.86 (m, 3H); 13C NMR (300 MHz,CDCl3) 207.79, 169.64, 168.80, 166.61, 162.51, 157.47, 149.00,147.48, 141.96, 133.38, 129.95, 120.20, 119.99, 113.71, 113.47,111.9, 111.52, 76.4, 67.19, 60.33, 56.73, 51.28, 46.68, 44.13, 38.96,36.62, 32.56, 31.74, 27.51, 26.41, 24.92, 23.61, 21.09, 8.77; MS(calcd. 640.3360, ES�): 641.3446 (M�H)�.

(S)-((R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(3-hydroxypropylamino)-2-oxoethoxy)phenyl)propyl) 1-(2-phenylacetyl)piperidine-2-carboxy-late, AP-1780 alcohol (Fig. S1C). Starting material, 2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-((S)-1-(2-phenylacetyl)piperidine-2-carbonyloxy)propyl)phenoxy)acetic acid, was prepared with thereported synthetic procedure (6).

The starting acid (202 mg, 0.352 mmol, 1 eq.) and CDI (68.4mg, 0.422 mmol, 1.2 eq.) were dissolved in anhydrous DMF (4mL) under nitrogen. The mixture was stirred at room temper-ature for 15 min to activate the acid. 3-Amino-1-propanol (134.0�L , 132 mg, 1.758 mmol, 5.0 eq.) was added to the solution andthe reaction was maintained at room temperature for 1 h. Thereaction mixture was then concentrated and purified by auto-mated flash chromatography to afford the pure product (161.1mg, 72%). 1H NMR (300 MHz, CDCl3) � 7.47 (s, 1H), 7.24 (m,7H), 6.85 (t, 2H, J � 6.4), 6.77 (d, 2H, J � 5.6), 6.65 (t, 3H, J �10.2), 5.70 (dd, 1H, J � 5.1, 8.2), 5.45 (s, 1H), 4.44 (d, 2H, J �4.0), 3.83 (d, 7H, J � 1.4), 3.74 (d, 3H, J � 12.1), 3.66 (s, 1H),3.56 (dd, 2H, J � 4.7, 10.3), 3.43 (m, 3H), 3.17 (d, 1H, J � 10.4),2.91 (s, 1H), 2.84 (s, 1H), 2.55 (dd, 3H, J � 5.8, 14.5), 2.29 (d, 1H,J � 13.2), 2.16 (d, 1H, J � 5.7), 2.02 (s, 2H), 1.66 (d, 6H, J � 7.4),1.25 (dd, 3H, J � 8.0, 15.1); 13C NMR (300 MHz, CDCl3) 171.18,169.76, 168.62, 162.53, 149.00, 147.47, 142.19, 134.72, 133.46,129.84, 128.67, 126.77, 120.22, 119.76, 113.36, 113.22, 111.92,111.57, 75.97, 67.17, 60.12, 55.96, 52.16, 44.01, 40.95, 38.09, 36.89,31.44, 31.29, 26.68, 25.06, 20.76; MS (calcd. 632.3098, ES�):633.3164 (M�H)�.

(S)-((R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(3-hydroxypropylamino)-2-oxoethoxy)phenyl)propyl) 1-(3,3-dimethyl-2-oxopentanoyl)pyrroli-dine-2-carboxylate, ProAP-1497 alcohol (Fig. S1D). Starting material,2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-((S)-1-(3,3-dimethyl-2-oxopentanoyl)pyrrolidine-2-carbonyloxy)propyl)phenoxy)aceticacid, was prepared with the reported synthetic procedure (6).

The starting acid (137 mg, 0.240 mmol, 1 eq.) and CDI (46.7mg, 0.288 mmol, 1.2 eq.) were dissolved in anhydrous DMF (2mL) under nitrogen. The mixture was stirred at room temper-ature for 15 min to activate the acid. 3-Amino-1-propanol (92.0�L, 90.0 mg, 1.20 mmol, 5.0 eq.) was added to the solution andthe reaction was maintained at room temperature for 1 h. Thereaction mixture was then concentrated and purified by auto-mated flash chromatography to afford the pure product (114.6mg, 76%). 1H NMR (300 MHz, CDCl3) � 7.34 (d, 1H, J � 5.8),7.26 (m, 1H), 6.92 (m, 2H), 6.78 (m, 2H), 6.67 (m, 2H), 5.70 (m,1H), 4.61 (m, 1H), 4.50 (s, 2H), 3.81 (dd, 6H, J � 4.6, 18.8), 3.62(m, 3H), 3.49 (dt, 5H, J � 5.6, 18.2), 2.92 (d, 1H, J � 1.4), 2.85(d, 1H, J � 1.2), 2.54 (m, 2H), 2.21 (m, 3H), 2.00 (m, 4H), 1.68(qq, 4H, J � 6.8, 13.3), 1.20 (t, 4H, J � 7.5), 1.13 (s, 1H), 0.93(s, 1H), 0.82 (dd, 2H, J � 6.8, 7.6); 13C NMR (300 MHz, CDCl3)207.00, 171.42, 168.86, 163.4, 162.52, 148.95, 147.41, 142.02,133.54, 129.85, 120.19, 119.79, 113.48, 113.29, 111.97, 111.5,76.02, 67.21, 59.97, 58.51, 55.95, 47.21, 46.53, 38.04, 36.41, 32.61,31.64, 31.11, 29.01, 24.85, 8.74; MS (calcd. 626.3203, ES�):627.3299 (M�H)�.

2-(3-((R)-3-(3,4-dimethoxyphenyl)-1-((S)-1-(2-phenylacetyl)pyrroli-dine-2-carbonyloxy)propyl)phenoxy)acetic acid, ProAP-1780, (*) (Fig.S1E). Starting material, (R)-tert-butyl 2-(3-(3-(3,4-dimethoxy-phenyl)-1-hydroxypropyl)phenoxy)acetate, was prepared by thereported procedure1. Starting material (S)-1-(2-phenylac-etyl)pyrrolidine-2-carboxylic acid was prepared with commercialcompound proline reacting with phenylacetyl chloride as re-ported (7).

To alcohol (241 mg, 0.599 mmol, 1.0 eq.), the acid (154 mg,0.659 mmol, 1.1 eq) EDC (137.4 mg, 0.719 mmol and 1.2 eq) andDMAP (8.05 mg, 0.066 mmol, 0.11 eq) were dissolved inanhydrous CH2Cl2 (10.0 mL) at room temperature under nitro-gen. The reaction was stirred for 2.5 h. The crude reactionmixture was extracted with H2O, dried over MgSO4, and con-centrated before being purified by automated chromatographyto afford the tert-butyl-protected intermediate as colorless oil.The intermediate compound is directly dissolved in CH2Cl2 (9mL) at 0 °C before TFA (3.0 mL) was added. The reaction wasstirred for 30 min at 0 °C and 1 h at room temperature. The crudeproduct was obtained by concentrating the reaction mixtureunder vacuum, which was further purified by automated flashchromatography to afford the pure product (254.7 mg, 76% 2steps). 1H NMR (300 MHz, CDCl3) � 10.63 (s, 1H), 7.12 (d, 6H,J � 9.7), 6.72 (dt, 7H, J � 23.8, 32.4), 5.63 (s, 1H), 4.55 (s, 1H),4.39 (s, 2H), 3.74 (s, 6H), 3.62 (s, 2H), 3.45 (d, 3H, J � 33.0), 2.43(s, 2H), 1.96 (d, 7H, J � 56.1); 13C NMR (300 MHz, CDCl3)171.25, 170.83, 157.96, 158.12, 148.94, 147.37, 141.88, 133.98,133.71, 129.59, 128.96, 128.68, 126.93, 120.27, 119.53, 114.51,112.16, 112.02, 111.53, 77.6, 65.19, 59.35, 55.98, 47.56, 41.69,38.07, 31.19, 29.36, 24.74; MS (calcd. 561.2363, ES�): 562.2495(M�H)�.

(S)-((R)-3-(3,4-dimethoxyphenyl)-1-(3-(2-(3-hydroxypropylamino)-2-oxoethoxy)phenyl)propyl) 1-(2-phenylacetyl)pyrrolidine-2-carboxy-late, ProAP-1780 alcohol (Fig. S1F). The starting carboxylic acid (83mg, 0.148 mmol, 1 eq.) and CDI (28.8 mg, 0.177 mmol, 1.2 eq.)were dissolved in anhydrous DMF (2.0 mL) under nitrogen. Themixture was stirred at room temperature for 15 min to activatethe acid. 3-Amino-1-propanol (56.5 �L, 0.739 mmol, 5.0 eq.) wasadded to the solution and the reaction was maintained at roomtemperature for 1 h. The reaction mixture was then concentratedand purified by automated flash chromatography to afford thepure product (85.3 mg, 93%). 1H NMR (300 MHz, CDCl3) � 7.99(s, 1H), 7.59 (s, 1H), 7.25 (m, 6H), 6.96 (s, 1H), 6.87 (d, 1H, J �7.8), 6.77 (t, 2H, J � 6.6), 6.68 (d, 2H, J � 5.3), 5.72 (m, 1H), 4.62(m, 1H), 4.46 (s, 2H), 4.25 (m, 1H), 3.84 (s, 6H), 3.76 (m, 1H),3.69 (s, 2H), 3.64 (s, 1H), 3.45 (m, 6H), 3.29 (s, 1H), 2.93 (s, 2H),

Ong et al. www.pnas.org/cgi/content/short/0900191106 3 of 15

2.86 (s, 2H), 2.55 (m, 2H), 2.19 (m, 3H), 1.96 (m, 5H), 1.61 (s,2H); 13C NMR (300 MHz, CDCl3) 171.61, 170.00, 168.75, 162.52,148.98, 147.43, 142.22, 134.24, 133.63, 129.65, 128.95, 128.61,126.85, 120.21, 119.51, 113.44, 112.88, 111.99, 111.53, 77.56,67.15, 60.14, 59.13, 55.97, 47.43, 41.85, 39.89, 36.97, 31.25, 29.38,24.83, 21.2; MS (calcd. 618.2941, ES�): 619.3025 (M�H)�.

2-(4-(4-(4-fluorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenoxy)-N-(3-hydroxypropyl)acetamide, SB202190 alcohol. Starting materialSB202190 (4-(4-(4-f luorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenol) was purchased from Sigma (cat# S7067). Thestarting compound was subjected to 3 steps of chemical reactionswithout purification at intermediate steps (Step1 and 2)

Fig. S1G. Step 1: SB202190 (25.0 mg, 0.075 mmol, 1 eq.) wasdissolved in anhydrous THF (0.5 mL) and K2CO3 (209 mg, 1.509mmol, 20 eq.) was added at room temperature. The solutionturned deep red upon K2CO3 addition before tert-butyl 2-bro-moacetate (13.4 �L, 0.091 mmol, 1.2 eq.) was added. Thereaction was stirred at room temperature for 48 h and the crudereaction mixture was filtered and concentrated. The crudeintermediate compound tert-butyl 2-(4-(4-(4-f luorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenoxy)acetate obtained wasused directly for the next step without further purification.

Fig. S1H. Step 2: The crude tert-butyl 2-(4-(4-(4-f luorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenoxy)acetate intermedi-ate was dissolved in CH2Cl2 (1.0 mL) and TFA (0.3 mL) wasadded. The reaction was stirred at room temperature for 1 h. Thereaction mixture was concentrated and the crude product 2-(4-(4-(4-f luorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenoxy)acetic acid was used directly in the final step without furtherpurification.

Fig. S1I. Step 3: The crude 2-(4-(4-(4-f luorophenyl)-5-(pyridin-4-yl)-1H-imidazol-2-yl)phenoxy)acetic acid intermediate wasdissolved in anhydrous DMF (2.0 mL) and CDI (25.0 mg, 0.154mmol, 2.05 eq.) was added to activate the acid at room temper-ature. After 15 min, 3-amino-1-propanol (48.2 mg, 0.642 mmol,8.5 eq.) was added to the solution and the reaction was stirredat room temperature for 2 h. The crude reaction mixture wasconcentrated and purified by automated flash chromatographyto afford the pure product (23.8 mg, 71%, 3 steps). 1H NMR (300MHz, CD3OD) � 8.45 (d, 2H, J � 5.7), 8.05 (s, 2H), 7.99 (m, 2H),7.55 (m, 4H), 7.22 (t, 2H, J � 8.8), 7.14 (d, 2H, J � 8.9), 4.92 (s,13H), 4.60 (s, 2H), 3.63 (m, 8H), 3.41 (t, 2H, J � 6.8), 3.30 (m,9H), 2.89 (s, 1H), 1.76 (m, 9H), 1.30 (s, 1H); 13C NMR (300 MHz,CDCl3) 170.87, 167.38, 163.93, 162.75, 160.13, 149.83, 149.16,132.21, 132.10, 129.18, 128.75, 124.27, 117.09, 116.8, 116.33,68.34; MS (calcd. 446.1754, ES�): 447.1845 (M�H)�.

(�)-K252a alcohol derivative, (Fig. S1J). The starting material (�)-K252a was purchased from BioMol International L.P. (catalog#:EI152). (�)-K252a (25.0 mg, 0.043 mmol, 1.0 eq.) and lipaseacrylic resin (66 mg) from Candida antarctica (Sigma cat#L4777) were mixed in ethanolamine (4.0 mL, 1545 eq.) at 40 °Cunder nitrogen. The reaction was stirred overnight, filtered,washed with MeOH, concentrated and taken up with saturatedNH4Cl, and then extracted with EtOAc. The organic layer wasdried over Na2SO4, concentrated and purified by automatedflash chromatography to afford the pure product (19.5 mg,92%). 1H NMR (300 MHz, THF-d8) 1H NMR (300 MHz, THF)� 8.46 (d, 1H, J � 7.9), 7.11 (m, 2H), 6.78 (d, 1H, J � 8.2), 6.53(t, 2H, J � 7.7), 6.42 (t, 1H, J � 7.4), 6.33 (t, 1H, J � 7.5), 6.17(dd, 1H, J � 4.8, 7.3), 4.64 (s, 4H), 4.08 (q, 2H, J � 17.2), 2.81(d, 2H, J � 5.0), 2.55 (m, 2H), 2.38 (s, 1H), 1.35 (s, 3H); 13C NMR(300 MHz, CDCl3) 171.19, 170.90, 139.57, 136.43, 131.50, 128.09,125.59, 124.32, 123.47, 122.44, 119.79, 115.86, 113.85, 106.8,

99.31, 84.44, 84.40, 64.88, 46.90, 44.61, 41.86, 23.64; MS (calcd.496.1747, ES�): 497.1820 (M�H)�.

3-(1-(3-hydroxypropyl)-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione, RO31–7549 alcohol derivative (Fig. S1K). The prep-aration of RO31–7549’s alcohol derivative follows a procedurereported in ref. 8.

General Procedure for Immobilization of Bait Molecules.Fig. S1L. Bio-Rad Affi Gel 102, an agarose-based gel with a6-atom hydrophilic amino-terminated arm.Solid-phase (beads) preparation. The solid-phase beads used in smallmolecule immobilization and affinity chromatography is Affigel102 (Bio-Rad) with a loading level of 12 �mol/mL suspension(Fig. S1). The bead suspension (1.0 mL) was transferred to a 2.0mL Eppendorf tube and washed with H2O (3 � 1.5 mL) andDMF (3 � 1.5 mL). The beads were then suspended in anhy-drous DMF (0.5 mL).

Fig. S1M. Example of bait molecule activation (RO-31-7549).2) Bait molecule activation (Fig. S2): The bait molecule (10

�mol) was dissolved in 200 �L of anhydrous acetonitrile (orDMF). DSC (7.69 mg, 30 �mol, 3 eq.) was dissolved in 400 �Lof anhydrous acetonitrile (or DMF) and was added to the baitmolecule solution before TEA (5.55 �L, 4.05 mg, 40 �mol, 4 eq.)was added. The reaction solution was stirred at 50 °C for 1 h andthe activation efficiency was monitored by LC-MS.

Fig. S1N. Example of immobilization of activated bait molecules(RO-31-7549).Bait molecule immobilization (Fig. S3). Different bait compounds givedifferent activation efficiencies, therefore the volume of activa-tion solution added to the beads was calculated individually. Forexample, when the LC-MS indicated 85% of bait molecules wasactivated, for 6.25% loading level, 53.4 �L of activation solution(contains 0.75 �mol of activated bait molecule) was added to thebeads suspension; for 12.5% and 25.0% loading levels, 106.8 �Land 213.6 �L were added to the beads respectively. After addingthe activation solution, the suspension was vortexed at roomtemperature for 1 h and the depletion of free activated baitmolecule was monitored by LC-MS.Work-up. After the immobilization, the vials were centrifuged, thesupernatant was removed and the beads were washed with DMF(3 � 2 mL)and H2O (3 � 2 mL). The beads were subsequentlysuspended in 1� PBS (0.8 mL) and stored at 4 °C before use.

SI MaterialsSPR Binding Assays.

FKBP1A and MTAP to Immunophilin ligands.The binding of Immunophilin (IPL) ligands to FKBP1A was

measured by surface plasmon resonance. The observed responserates were too fast to be analyzed in kinetic terms (ka, kd).Affinity constants (KD) were derived from equilibrium bindingmeasurements. The values range from 26 nM to 44 �M (Fig. 5A).

Sensor Preparation. The FKBP12 surface plasmon resonanceassay was performed on a Biacore S51 instrument using BiacoreCM5 sensor chips. GST capture kit, ethanolamine, N-ethyl-N�-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuc-cinimide (NHS) and P-20 surfactant were all obtained fromBiacore Inc. Immunophilin igands were synthesized followingthe general procedure outlined by Holt. The surface of thesensor chip was conditioned using alternating 1 min injections(90 �L/min flow rate) of 10 mM glycine pH 2.2 and 50 mMNaOH (repeated 3 times). The surface carboxyl groups wereactivated with 1:1 0.4M EDC/0.1M NHS. A 25 �g/mL solutionof anti-GST inl acetate buffer pH 5.5 was flowed for 10 min ata rate of 5 �L/min over spots 1 and 2. The remaining NHS-ester

Ong et al. www.pnas.org/cgi/content/short/0900191106 4 of 15

groups on the sensor surface were quenched with a 7 mininjection of 1 M ethanolamine. Recombinant GST (10 �g/mLsolution in PBS-P pH 7.4) was captured on spot 1 followed byFKBP12-GST fusion protein (50 �g/mL solution in PBS-P pH7.4) on Spot 2. Table 1 contains information on sensor prepa-ration for each day’s assay.

Data Analysis. All data were analyzed using T100 Evaluation andScrubber software. Data were double reference subtracted. Thesteady state affinity constant (KD) for each ligand was derivedfrom a plot of Req against concentration. The plot was then fittedto a general steady state model. Table 2 contains KD valuesderived from equilibrium binding measurements.

Accompanying Text for Fig. S2: Interpretation of Bead Control Data.In analysis of SILAC data, a common practice is to normalize allquantified protein ratios to account for a systematic error madewhen combining protein lysates. This normalization is oftendone by determining dividing each protein’s ratio by the medianratio for the group of proteins that do not exhibit a significantchange in SILAC ratios. However, in the case of BC data (Fig.S2 A), this is not appropriate as ratio distributions shift consid-erably depending on the amount of compound loaded. Thedistributions of protein ratios itself is dramatically different in25% loaded beads (Fig. S2C). We analyzed lysates mixed one-to-one without any affinity enrichment to determine the mixingratio empirically and found this to be very close to 1 (1.0 � 0.15)in our experiments. This determination, in addition to gelvisualization, indicates that the effect of compound bead loadingin each BC experiment on SILAC ratio distributions was in factan accurate representation of protein binding specificities. How-ever, this makes data interpretation much less direct. We foundk-means clustering of data from multiple bead loadings to beuseful for this purpose (Fig. S3)

Accompanying Text for Fig. S3. We compared datasets generatedfrom 3 loading levels (6, 12 and 25%) of the kinase inhibitorRo-31-7549 with k-means clustering (Euclidean distance, num-ber of clusters: 3 through 7, only 3-cluster data shown), moni-toring membership of proteins within the modeled distributionsacross the different load levels (Fig. S3A). Known targets likePRKCD, PRKCA, GSK3B, ADK, CAMK2D, and NQO2among others, dominated a cluster of 42 proteins, characterizedby consistently high SILAC ratios irrespective of compoundloading levels, whereas 2 clusters comprising the majority of theproteins had ratios indicating increased binding to either EtOH-beads or small molecule-loaded beads (Cluster 1: 132 EtOH-

bead specific; Cluster 2: 269 SM-bead specific). We analyzedun-enriched whole cell lysates and compared protein abundancesin these samples to those identified in our pull-down experi-ments. Proteins in clusters 1 and 2 were significantly differentfrom whole cell lysates (Fig. 3B, Kolmogorov–Smirnov test, Pvalue: � 2.2 � 1016), whereas the cluster of target proteins hada K-S P value of 0.0026, also indicating that clusters 1 and 2 weremostly dominated by abundant proteins. Indeed, over half of thecluster 3 target proteins were not identified at all in the analysisof whole cell lysate, likely because they were not among the top�2000 proteins in abundance. We performed the same analysisusing the whole set of proteins identified in the SC experiment(Fig. S3C) and find the majority of proteins identified in SCexperiments are abundant proteins that were also identified inunenriched lysate.

Accompanying Text for Fig. S4. We compared the list of targetsidentified in SC and BC experiments and found that bothexperimental formats were effective at enriching known targets.We observed some proteins with high small molecule (SM)-specific SILAC ratios in BC experiments but ratios close to 1 inSC experiments, and hence, were nonspecific (for e.g., VDAC2,GSK3B, NQO2 at 10� SC, Fig. S4A). Using an orthogonalapproach with recombinant GST-fusion proteins and Westernblot analysis, we found that the GST-western experiments werein complete agreement with SILAC ratio data (Fig. S4B).Proteins identified in the BC experiment were indeed enrichedby small molecule affinity matrices, but soluble competition wasmost effective with the primary target PRKCD and not otherproteins, and depended on the levels of soluble competitor used.In applying a range of soluble competitor amounts in other SCexperiments, we made the general observation that our ability toidentify targets improved with increasing levels of soluble com-petition (Fig. 4).

Accompanying Text for Dataset S1. Dataset S1 contains multipleworksheets that summarize soluble competitor (SC) experimentsfor kinase inhibitors and the IPL series. The k252a and Ro-31-7549 tables are summarized across multiple experiments tocombine FDR values for each protein into a single summarystatistic. This was calculated as: 10[(w.f.) � log(local FDR)], with thetotal of weighting factors (w.f.) equaling 1. W.f. for differentexperiments were chosen heuristically and we provide 2 alternatesets of w.f. as examples. We chose w.f. that would raise thecontribution of higher SC levels or remove the influence ofexperiments that may have been affected by compound precip-itation (for e.g., Ro-31-7549 at 100� SC). The choice of w.f. doesnot dramatically change the list of targets.

1. Ong SE, Mann M (2006) A practical recipe for stable isotope labeling by amino acids incell culture (SILAC). Nat Protoc 1(6):2650–2660.

2. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment,pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc2(8):1896–1906.

3. Efron B, Tibshirani R (2002) Empirical bayes methods and false discovery rates formicroarrays. Genet Epidemiol 23(1):70–86.

4. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinasecomplement of the human genome. Science 298(5600):1912–1934.

5. Christopher SA, Diegelman P, Porter CW, Kruger WD (2002) Methylthioadenosinephosphorylase, a gene frequently codeleted with p16(cdkN2a/ARF), acts as a tumorsuppressor in a breast cancer cell line. Cancer Res 62(22):6639–6644.

6. Keenan T, et al. (1998) Synthesis and activity of bivalent FKBP12 ligands for theregulated dimerization of proteins. Bioorg Med Chem 6(8):1309–1335.

7. Boto A, De Leon Y, Gallardo JA, Hernandez R (2005) Synthesis of alkaloid analoguesfrom alpha-amino acids by one-pot radical decarboxylation/alkylation. (Translatedfrom English) Eur J Org Chem (16):3461–3468 (in English).

8. Faul MM, Winneroski LL, Krumrich CA (1998) A new, efficient method for the synthesisof bisindolylmaleimides. (Translated from English) J Org Chem 63(17):6053–6058 (inEnglish).

Ong et al. www.pnas.org/cgi/content/short/0900191106 5 of 15

R O H

O

OONN

O

OO

OR O O

O

N

O

O

H 2 N

R O NH

O

A ff ige l-10 2im m o b il iz at i ona ct i v at io n

HN

Im m u n o p h ilin L ig an d s

OO

NM e

N

HO

R O -31 -75 49

NH

NO

N

F

O

HNO H

S B -2 021 90

HN O

NNO

M e

O H

O

NHHO

K 25 2a*

A

BM eO

M eO OOO

NO

OO

H N O H

M eO

M eO OOO

NO

OO

O H CD I, D M F

3-am ino -1-p ropanol

M eO

M eO O

OON

OO

H N O H

CD I, D M F

3-am ino -1-p ropanol

M eO

M eO O

OON

OO

O H

C

C DI, DM F

3-am ino -1 -p ropano l

M eO

M eO ON H

OON

OO

O

O H

M eO

M eO OO H

OON

OO

O

DM eO

M eO O OtB u

OHO

NO

OH

OD M A P , C H 2 C l2

E D C

M eO

M eO O OH

OON

OO

E

C DI, DM F

3-am ino -1 -p ropano l

M eO

M eO OOO

NO

O

HN O H

M e O

M e O OOO

NO

O

O HF

F

N

HN

N

O H

F

N

HN

N

O O

Ot er t -bu ty l 2 -b rom oace ta te

K 2CO 3 in TH F

G

F

N

HN

N

O O

O

F

N

HN

N

O O

OHT F A in CH 2C l2

H

3-am ino -1-propano l

CDI, D M F

F

N

HN

N

O O

O H

F

N

HN

N

O O

H N

O HI

N N

HN

O

M eO 2CO H

O

N N

HN

O

O H

O

O

HN

HO

lipase acry lic resin

e thano lam ine , 40 ºC

J

H N

O

O N

NO H

K

ONH

OH2 N

L

NO O

NO

O

O

O

O

H N

O

O N

N

OH H N

O

O N

NO

O N

O

O

O

act iva t ion

M

H N

O

O N

NO

O N

O

O

O H N

O

O N

NO

O

ONH

OH N

ONH

OH 2 N

N

O O

N

O

R

O

O

HN

M e O

M e O

n = 0,1

O H

Fig. S1. Generation of affinity matrices for small-molecule pull-down experiments. (A) A general scheme of the coupling reaction of a small molecule containinga functionalizable handle (OH in this case) to a solid support (Affigel-102) is shown. The chemical structures of immunophilin ligands and kinase inhibitors usedare shown. (B–N) Chemical reaction schemes to match SI Methods.

Ong et al. www.pnas.org/cgi/content/short/0900191106 6 of 15

AP1497 25%

MW

188

98

62

49

38

28

1714

6

EtOHbead25%

AP149725%

AP1497 12%EtOHbead12%

AP149712%

AP1497 6%EtOHbead6%

AP14976% MW

188

98

62

49

38

28

1714

6

A B C

Ro 31-7549 6% Ro 31-7549 12.5% Ro 31-7549 25%

Low Stringency

Ro 31-7549 6% Ro 31-7549 12.5% Ro 31-7549 25%

High Stringency

Low Stringency

High Stringency

AP1497 Pro-AP1497 AP1780 Pro-AP1780

AP1497 Pro-AP1497 AP1780 Pro-AP1780

Immunophilin Ligand SeriesRo-31-7549 bead loading 6,12,25%D

Fig. S2. (A–C) Gel visualization, SILAC ratio distribution plots for Immunophilin ligand AP1480 at 3 compound load levels (6%, 12%, 25%). For each load level,unmixed SILAC pull-downs were resolved on a 4–12% gradient Bis-Tris gel with each bead type and lysate loaded in a separate gel lane. A parallel set ofpull-downs was mixed and loaded on the gel for quantitative MS analysis. Log2 SILAC ratios from that set is plotted as a histogram (Upper) or plotted with SILACratios sorted in descending order (Lower). (D) Modeled distributions of SILAC ratios for bead control experiments for different molecules. The different ratiosdistributions are accurate representations of the binding characteristics of the affinity matrices (see also Fig. S2 A–C). The variability depends on variousparameters such as the small molecule used, loading density of small molecules on bead, and stringency of wash buffer used. High stringency washing reducedbackground interactions to a degree. Target proteins were largely unaffected by high stringency washing, comparing LS and HS experiments is therefore a usefulmethod for identifying SM-specific targets. However, very abundant proteins that bound to SM-beads were still identified with high SILAC ratios, thereby stillrequiring cross-experiment comparisons to avoid this bias (see Fig. S3). Because log2 ratio values for BC experiments in general displayed visually separablepopulations, we modeled these data using mixtures of t-distributions and selected the optimal number of components using the Bayesian information criterion(BIC). We note that the data were not well modeled by Gaussian distributions. Indeed, BIC analysis indicates that the data are best described by at least 2t-distributions. The plots display the number of components corresponding to the lowest BIC score.

Ong et al. www.pnas.org/cgi/content/short/0900191106 7 of 15

-50

5

Ro Loading Lev el

log2

SIL

AC

ratio

-50

5

Ro Loading Lev el

log2

SIL

AC

ratio

-50

5

Ro Loading Lev el

log2

SIL

AC

ratio

Cluster 2:269 SM bead binders

Cluster 1: 132 EtOH bead binders

Cluster 3: 42 Ro-31-7549 targets

6 12 25 6 12 25 6 12 25

A

RankRo25

RankEtOH RankTargetRankSM

Cluster 1 Cluster 2 Cluster 3

1.503e-11 < 2.2e-16 3.083e-10

< 2.2e-16

22 of 42 (52%)not identifiedin whole lysate

88 of 269 (33%)not identifiedin whole lysate

29 of 132 (22%)not identifiedin whole lysate

Ro25_10x_SC

618 identified,218 not identified inwhole lysate

1

2087

1

2087

1

2087

1

2087

B C

Most abundant

Leastabundant

Ranked byProtein XICs

Most abundant

Leastabundant

Ranked byProtein XICs

k-means clustering of Ro-31-7549 BC data across three loading levels

Ro-31-7549 BC k-means clusters mapped to HeLaS3 whole cell lysates Ro-31-7549 SC proteins mapped to HeLaS3 whole cell lysates

Fig. S3. k-means clustering and pull-down enrichment of proteins. (A) BC protein ratios from 3 loading levels of Ro-31-7549 were clustered into 3 clusters withk-means. Known and putative targets were found exclusively in cluster 3, characterized by high SILAC ratios across all 3 load levels. (B) Evaluating affinityenrichment of 3 bead binding specificities (k-means clusters) in reference to ranked protein abundance from unfractionated whole cell lysate by Kolmogorov–Smirnov. Specific bias toward abundant proteins was observed with weak affinity EtOH- or SM-bead binders (Clusters 1 and 2, k-s p value �2 � 1016). Targetproteins in cluster 3 show much weaker significance (p value 0.0026) and the percentage of proteins identified in affinity pull-downs but undetected in wholecell lysate is also highest in cluster 3. (C) The mapping of proteins in whole cell lysates to the RO25�10��SC dataset also shows that the SC dataset containsabundant proteins from lysates. However, the SC experimental design allows identification of specific targets because abundant proteins have ratios close toone-to-one.

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RO Sol. comp - 0.1x 0.5x 1x

Pull-downs Directinput

SILAC ratios Bead Ctrl

Soluble Comp.

0.1x 1x

PRKCD

GSK3B

PRKCD

GSK3B

>50

10 1.1 1.4

4.5 15

NQO2 45 0.97 0.96

A

B

PRKCD

GSK3B

NQO2

VDAC2

GST

-

VDAC2 7.2 0.94 0.92

Fig. S4. Validation of SILAC pull-down data with recombinant proteins. (A) Table of SILAC ratios for proteins identified as specific protein interactors to thekinase inhibitor Ro-31-7549. (B) Validation of SC data. Equimolar amounts of purified GST-fusion proteins were incubated overnight with Ro-31-7549 with orwithout soluble competition. This experiment shows that although many proteins may be identified as specific binders in BC experiments (Table in Fig. 4), someof these (GSK3B, NQO2, VDAC2) show diminished selectivity toward soluble forms of the small molecule. The negative controls FKBP12 and GST-alone show nobinding to the solid phase matrix.

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G ene sym bo lk252a pc12 B C

k252a h1299 B C

k252a H eLa B C

k252a H eLa S C

S B 202190 H eLa B C

S B 202190 H eLa S C

R o31-7549 H eLa B C

R o31-7549 H eLa S C

A A K 1A D KA U R K AB M P 2KC A M K 2AC A M 2K BC A M K 2DC A M K 2GC D C 2C D C 2L5C D C 42B P BC D K 2C D K 4C D K 5C D K 6C D K 7C D K 9C LK 1C LK 4C H E K 1C ITC R K R SC S KC S N K 2A 1C S N K 2A 2C S N K 1DD A P K 2D E KE IF 2A K 2E IF 2A K 4FERF R A PG A KG S K 3AG S K 3BH U N KIR A K 4LIM K 1M A P 2K 1M A P 2K 2M A P 2K 3M A P 2K 4M A P 2K 5M A P 2K 6M A P 3K 9M A P 3K 11M A P 3K 15M A P 4K 4M A P K 1M A P K 3M A P K 8M A P K 9M A P K 10M A P K 14M A P K A P K 2M A R K 1M A R K 2M A R K 3M A S T 1M IN K 1M S T 4/R P 6-213H 19.1M YLKP A K 1P A K 4P C T K 1P C T K 2P D P K 1P D X KP H K G 2P K N 1P K N 2P R K A A 1P R K A C AP R K A C BP R K A G 1P R K A R 1P R K A R 2AP R K A R 2BP R K C AP R K C DP R K C IP R K D 1P R K D 2P R K D 3P R K D CP R K R AP T K 2R IO K 2R IP K 2R IP K 4R O C K 2R P S 6K A 1R P S 6K A 3S K P 2S LKS R P K 1S T K 10S T K 17AS T K 3S T K 4T B K 1T G FB R 1T G FB R 3T P 53R KT T KU LK 1U LK 3V R K 2YE S 1T ota l, S pec ific 40 42 47 56 9 8 44 21

>2 peps, bu t no t s ign ifican t>2 peps, s ign ifican tnot detected

A

0 5 10 15 20 25 30 35 400

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Number of Kinases

Pre

cisi

on

SCBC

B

Fig. S5. (A) Accuracy of kinase identification for K252a 100� SC and BC experiments. Proteins are sorted by their average log2 ratio (high to low) across replicateexperiments. Protein kinases (4) are used as a true positive set and at each rank the number of inferred kinases is plotted against the precision, which correspondsto the percentage of kinases among all inferred targets at a given threshold. (B) Kinases identified from kinase inhibitor experiments. Kinases identified in BCand SC experiments with k252a, Ro 31–7549, and SB202190 are summarized. Red indicates kinase was identified with �2 peptides, and with a significant SILACratio. Gray indicates protein was identified with �2 peptides but lack of specificity for the small molecule.

Ong et al. www.pnas.org/cgi/content/short/0900191106 10 of 15

MTAP Ratio in Bead Contro l Low /H igh S tringencies

0

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tio

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A

B

MTAP ratio and intensity plot in SC

02468101214161820

SIL

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ra

tio

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25B illions

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rote

in X

IC

10x H/L ratio100x H/L RatioTotal intensity 10xTotal intensity 100x

AP1497

ProAP-1

497

AP1780

ProAP-1

780

Fig. S6. MTAP is a protein binder to the immunophilin ligand series. (A) MTAP ratios determined from bead control experiments with the IPL series. MTAP isidentified as a specific binder to the IPL ligands in both low and high stringency bead control experiments. (B) MTAP ratios in SC experiments overlaid with plotsof total extracted ion intensities (XIC) for the protein. Although the SILAC ratio is largest in the SC experiment with AP1780, the total protein XIC indicates thatthe Pro-AP1780 affinity matrix enriched the most MTAP across the IPL series, suggesting that the levels of soluble competitor used in this experiment (max: 100�)was insufficient to compete with protein bound to the solid phase.

Ong et al. www.pnas.org/cgi/content/short/0900191106 11 of 15

-5

0

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-20 0 20 40 60 80 100 120

RU

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sTime (0 = Sample start)

12.3 nM

1000 nM

111 nM333 nM

37.0 nM

FKBP12 : AP1497 Sensogram

4.12 nM

2

6

10

14

18

0 2e-7 4e-7 6e-7 8e-7 1e-6

RU

Res

po

nse

ConcentrationM

KD = 25.7 nM

Steady State Affinity AP1497

-5

0

5

10

15

20

25RU

Res

po

nse

(0 =

base

line)

Time (0 = Sample start)

FKBP12 : AP1780 Sensogram

3.75 μμM

15.0 μμM

60.0 μμM30.0 μμM

7.50 μμ M

1.88 μμM

246810121416

0 1e-5 2e-5 3e-5 4e-5 5e-5 6e-5 7e-5

RU

Res

po

nse

ConcentrationM

KD = 5.17 μμMSteady State Affinity AP1780

-5

0

5

10

15

20

25RU

Res

po

nse

(0 =

base

line)

sTime (0 = Sample start)

0.156 μμM

10.0 μμM

0.313 μμM

2.50 μμM5.0 μμM

1.25 μμ M

0.625 μM

FKBP12 : Pro-AP1497 Sensogram

4

8

12

16

20

0 2e-6 4e-6 6e-6 8e-6 1e-5

RU

Res

po

nse

ConcentrationM

KD = 0.800 μμMSteady State Affinity Pro-AP1497

-5

0

5

10

15

20

25RU

Res

po

nse

(0 =

base

line)

sTime (0 = Sample start)

15.0 μμM

120 μμM

30.0 μμM

3.75 μ μM

60.0 μμM

7.50 μμM

FKBP12 : Pro-AP1780 Sensogram

1.88 μμM

02468101214

0 2e-5 4e-5 6e-5 8e-5

RU

Res

po

nse

ConcentrationM

KD = 43.8 μμMSteady State Affinity Pro-AP1780

MTAP-GST : Pro-AP1780 Sensogram

Steady-state affinity Pro-AP1780

MTAP-GST : AP1780 Sensogram

Steady-state affinity AP1780

MTAP-GST : AP1497 Sensogram

Steady-state affinity AP1497

MTAP-GST : Pro-AP1497 Sensogram

Steady-state affinity Pro-AP1497

0 10 20 30 40 50 60-10 70

1.2e-41e-4

0 10 20 30 40 50 60-10 70 0 10 20 30 40 50 60-10 70

Fig. S7. SPR binding curves for FKBP1A and MTAP with immunophilin ligands.

Ong et al. www.pnas.org/cgi/content/short/0900191106 12 of 15

Table S1. Biacore Steady State Affinity Data FKBP12

Date Sensor Chip FKBP-ligand Steady-state KD

5/21/07 1162163–2 AP1497 33.9 nMAP1497* 25.7 nMAP1780 14.7 �MAP1780* 5.17 �MPro-AP1497 566 nM

6/01/07 1162163–15 AP1780* 6.70 �MPro-AP1497 800 nMPro-AP1780 43.8 �M

*Ligands from different batch

Ong et al. www.pnas.org/cgi/content/short/0900191106 13 of 15

Table S2. SPR data of MTAP-GST with Immunophilin ligand series

IPL ligand KD equil KD kinetic ka, M1�s1 kd, s1

Pro-AP1780 18.0 � 2nM 12.4 � 1nM 4.29 � 106 0.053AP1780 1.55 � 2nM 2.43 � 4nM 1.86 � 107 0.045AP1497 2.82 � 3�M 1.9 � 1�M 50217.0 0.09315ProAP1497 8.54 � 7�M 20.0 � 9�M 3395.9 0.07208

Ong et al. www.pnas.org/cgi/content/short/0900191106 14 of 15

Other Supporting Information Files

Dataset S1 (XLS)

Table S3. List of tables in Dataset S1

Name of table SC level # experiments # replicates Comments

K252a Multiple Seven SC levels 2 per SC level SummarizedRo-31–7549 Multiple 2 bead loadings, five SC levels 2 per experiment SummarizedSB202190* Multiple T2 SC levels 10� , 100� 3 pooled per SCAP1497�10� (HoltHolt) 10� 1 2AP1497�100� (HoltHolt) 100� 1 2ProAP1497�10� (HoltPro) 10� 1 2ProAP1497�100� (HoltPro) 100� 1 2AP1780�10� (HoltBz) 10� 1 2AP1780�100� (HoltBz) 100� 1 2ProAP1780�10� (HoltDmg) 10� 1 2ProAP1780�100� (HoltDmg) 100� 1 2

Ong et al. www.pnas.org/cgi/content/short/0900191106 15 of 15