Supporting Information Ong et al. 10.1073/pnas.0900191106 SI Methods volume of DMSO is then added to 2 mg of heavy HeLaS3 as a 13 13 15 ␮ Materials and Reagents. L-arginine- C6 and L-lysine- C6 N2 control. 25 L of 50% of SM-bead is added to both light and were from Sigma Isotec. The cell culture media, Roswell Park heavy lysates in SC experiments. Memorial Institute-1640 (RPMI) deficient in arginine, lysine Affinity enrichments are incubated overnight (Ϸ16 h) on an and methioine, was a custom media preparation from Caisson end-over-end rotator at 4 °C. After incubation, the tubes are ϫ Laboratories. All other L-amino acids were obtained from spun at 1000 g on a benchtop centrifuge to pellet the beads. Sigma. Dialyzed serum was obtained from SAFC-Sigma. Trypsin The supernatant is aspirated, taking care to avoid disturbing the was from Promega and HeLaS3 and H1299 were a kind gift from beads. In BC experiments, beads are combined at the first wash Dr. James Bradner. A PC-12 rat pheochromocytoma cell line for subsequent washing steps. For SC experiments, each tube in stably expressing hErbB4 and GFP was generated with neomycin a set is washed with ModRIPA buffer at least once (twice with selection and 2 independent pcDNA3-neomycin (Invitrogen) high levels of soluble competition) to remove excess soluble constructs. Other cell lines were from ATCC. small molecule competitor. Beads from the 2 tubes were then be Antibodies against the following proteins were used: Anti- combined for later washing steps. In high stringency experi- FKBP1A and anti-MTAP (Abcam); anti-FKBP2 and anti- ments, a wash buffer (high stringency HS) containing ModRIPA FKBP5 R&D Systems); anti-FKBP4 (Bethyl Laboratories); anti- and 0.2% SDS was used to wash beads instead. After the third FKBP8 (US Biological); anti-FKBP10 (listed in the article as and final wash, beads are collected by spinning at 1000 ϫ g and FKBP9/10) (BD Transduction Laboratories). Secondary anti- the wash is aspirated leaving Ϸ20 ␮L of buffer in the tube. bodies were all purchased from Sigma. Anti-GST antibody was from GE Healthcare (Piscataway, NJ). All other reagents and 1D-SDS/PAGE and MS Analysis. Proteins enriched in SILAC affinity chemicals used were of the highest grade available. pull-downs were reduced and alkylated, on bead, in 2 mM DTT and 10 mM iodoacetamide respectively. One part LDS buffer SILAC Media Preparation and Cell Culture Conditions. We followed (Invitrogen) was added to 3 parts sample (including beads) and all standard SILAC media preparation and labeling steps as tubes heated to 70 °C for 10 min. Proteins were resolved on a described in ref. 1. Briefly, 15 mg/L of L-methionine was added 4–12% gradient 1.5 mm thick Bis-Tris gel with Mes running to base media according to standard formulations for standard buffer (Nupage, Invitrogen) and Coomassie stained (Simply RPMI. This base media was divided into 2 and either ‘‘light’’ Blue, Invitrogen). Gel lanes were excised into 6 pieces and then forms of arginine and lysine or ‘‘heavy’’ L-arginine-U-13C6 (87.2 further cut into 1.5 mm cubes. The gel pieces were further mg/L) and L-lysine-13C615N2 (155 mg/L) was added to generate destained in a solution containing 50% EtOH and 50% 50 mM the 2 SILAC labeling mediums. Each medium with the full ammonium bicarbonate, then dehydrated in 100% EtOH before complement of amino acids was sterile filtered through a 0.22 addition of sufficient trypsin (12.5 ng/␮L) to swell the gel pieces ␮M filter (Milipore). completely. An additional 100 ␮L of 50 mM ammonium bicar- HeLa S3 cells were grown in RPMI labeling media, prepared bonate was added before incubating at 37 °C overnight on a as described above, supplemented with 2 mM L-glutamine, and thermomixer (Eppendorf). Enzymatic digestion was stopped by 5% dialyzed FBS plus antibiotics, in a humidified atmosphere the addition of 100 ␮L of 1% TFA to tubes. A second extraction ␮ with 5% CO2 in air. Cells were grown for at least 6 cell divisions with 300 L of 0.1% TFA was combined with the first extract and in labeling media, initially growing in flasks but expanded into the peptides from each gel slice cleaned up on C18 StageTips (2). 2L spinner flasks on a magnetic stir plate to provide larger Peptides were eluted in 50 ␮L of 80% acetonitrile/0.1% TFA and cultures. dried down in a evaporative centrifuge to remove organic solvents. The peptides were then resuspended by vortexing in 7 Biochemical Purification with Small Molecule Affinity Matrices. Sep- ␮L of 0.1% TFA and analyzed by nanoflow-LCMS with an arate cultures of HeLaS3 cells SILAC labeled either with Agilent 1100 with autosampler and a LTQ-Orbitrap. Peptides L-arginine and L-lysine (light) or L-arginine-13C6 and L-lysine- were resolved on a 10 cm column, made in-house by packing a 13C6-15N2 (heavy) are lysed in ice-chilled ModRIPA buffer (low self-pulled 75 ␮m I.D. capillary, 15 ␮m tip (P-2000 laser based stringency buffer LS) containing 1% Nonidet P-40, 0.1% Na puller, Sutter Instruments) column with 3 ␮m Reprosil-C18-AQ deoxycholate, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5, beads (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) and protease inhibitors (Complete tablets, Roche Applied Sci- with an analytical flowrate of 200 nL/min and a 58 min linear ence, Indianapolis, IN). Lysates are vortexed intermittently while gradient (Ϸ0.57%B/min) from 0.1% formic acid in water to 0.1% chilled on ice for 10 min and clarified by spinning at 14,000 ϫ g. formic acid/90% acetonitrile. The run time was 108 min for a Protein concentrations of light and heavy lysates are estimated single sample, including sample loading and column recondi- with the Protein Assay Dye Reagent Concentrate (Bio-Rad, tioning. Hercules CA) and equalized. The protein concentrations of We used a MS method with a master Orbitrap full scan (60,000 lysates can vary between 1.7 to 2.2 mg/mL, affinity enrichments resolution) and data dependent LTQ MS/MS scans for the top are performed in lysate volumes between 1 mL to 1.4 mL in a 1.5 5 precursors (excluding z ϭ 1) from the Orbitrap scan. Each cycle mL microcentrifuge tube. was Ϸ2 secs long. MS raw files were processed for protein In bead control (BC) experiments, 2 mg of light HeLaS3 lysate identification and quantitation using extract࿝msn.exe (Thermo), is incubated with 25 ␮L of 50% slurry of EtOH-bead while 2 mg Mascot (Ver. 2.1.03 Matrixscience), and academic software, of heavy lysate is incubated with 25 ␮L of 50% small molecule DTASupercharge and MSQuant (CEBI, open-source http:// affinity beads (SM-bead). This is termed a ‘‘forward’’ label msquant.sourceforge.net). MS/MS peak lists in Mascot Generic experiment. In the ‘‘reverse’’ experiment, the lysates are Format were generated using extract࿝msn.exe and DTASuper- swapped for each bead type. In a ‘‘forward’’ soluble competitor charge and searched with Mascot using IPI human ver.3.32 experiment, the appropriate amount of small molecule (in (http://ebi.ac.uk) with a concatenated decoy database containing DMSO) is added to 2 mg of light HeLaS3 lysate. An equal randomized sequences from the same database. Common con- Ong et al. www.pnas.org/cgi/content/short/0900191106 1of15 taminants like BSA, trypsin etc. were also added to the database. FKBP10/FKBP9 Antibody Cross-Specificity Determined by Immunopre- Variable modifications used were oxidized methionine, argi- cipitation and MS (IP-MS). In Western blot analysis experiments to 13 13 15 nine- C6, lysine- C6 N2, and carbamidomethyl-cysteine was a validate our IPL ligand SILAC TargetID data, we found the fixed modification. The precursor mass tolerance used in the FKBP10 antibody (BD Transduction Laboratories, Cat. No. search was 15 ppm and fragment mass tolerance was 0.7 Da. 610648) yielded a strong band at Ϸ63 kDa in IPL ligand Proteins with a minimum Mascot score of 66 (at least 1 peptide pull-downs with AP-1480, ProAP-1480, and AP-1497. This was with score Ͼ66) and peptides with score Ͼ20 were quantified by not consistent with our SILAC data for FKBP10, but matched MSQuant. Only proteins with a minimum of 2 quantifiable the compound specificity profile for FKBP9. Because FKBP9 peptides were included in our dataset. The false positive rate for and FKBP10 are very similar in length, 570 aa and 582 aa protein identification is Ͻ1% and Ͻ5% at the peptide level, as respectively and are 58% identical at the amino acid level determined using the decoy database strategy. (BlastP), we hypothesized that the antibody used in our exper- iment was cross-reacting with both proteins. We evaluated this Statistical Analysis of SC Experiments. To model log2 protein ratio with an IP-MS experiment. We incubated 10 ␮g of antibody and values from SC experiments, we adapted the empirical Bayes 50 ␮L of a 50% slurry of Protein G beads with 2 mg of HeLaS3 framework developed by Efron (3) to compute the posterior lysate in our usual pull-down conditions. We used Protein probability that a ratio value arises from the null distribution. G-bead alone with lysate in a separate pull-down as a control. Briefly, by application of Bayes’s theorem, this quantity is Beads were washed, proteins reduced, alkylated, resolved on a Ϸ computed as Pr(Z ϭ 0͉X) ϭ [Pr(X͉Z ϭ 0)Pr(Z ϭ 0)]/Pr(X) where 1D-SDS/PAGE gel and the molecular mass bands between 60 Z is a binary variable taking the value of 1 if the protein is bound to 98 kDa were excised for MS analysis.