Copyright WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001. Supporting Information for Angew. Chem. Int. Ed. Z 18050 Protein Affinity Labeling Mediated by Genetically Encoded Peptide Tags Frank Amini, Thomas Kodadek*, Kathlynn C. Brown* I- Protein affinity labeling: GGH Ecotin (2 µl of a 75 µm solution in ddh 2 0) was incubated with Ni(OAc) 2 (1 µl of a 1.5 mm solution in ddh 2 0) for 15 minutes at room temperature. The sample was then diluted in crosslinking buffer (150 mm sodium phosphate and 150 mm sodium chloride, ph 7.0) to 12.5 µl. Biotin-tyramine (1.5 µl of a 2.5 mm solution in 10% dimethyl-sulfoxide) was then added to the solution. Finally, the reaction was initiated by the addition of MMPP (1 µl of a 1.5 mm solution in cross-linking buffer). The sample was incubated at room temperature for 2 minutes and subsequently quenched with 5 µl of loading buffer (0.24 M Tris, 8% SDS, 2.88 M β-mercaptoethanol, 40% glycerol, and 0.4% bromophenol blue). The final concentrations of GGH-Ecotin, Ni II, biotin-tyramine, and MMPP were 10 µm, 100 µm, 250 µm, and 100 µm, respectively. In some samples, free GGH (1 µl of a 1.5 mm 1
solution in ddh 2 0) was used instead of GGH-Ecotin. The final concentration of the Ni II -GGH complex in these samples was 100 µm. Finally, one or two other proteins were included in some samples. In such cases, the proteins were added immediately after the incubation of Ni(OAc) 2 with GGH-Ecotin. The proteins were then allowed to equilibriate at room temperature for 5 minutes before the sample was diluted in cross-linking buffer. The final concentrations of the added proteins (Trypsin, GST- Ubl, and Ub) were 20 µm. The same protocol was used for the affinity labeling of His 6 - Gal80, except that the final His 6 -Gal80 and Ni(OAc) 2 concentrations were 4 µm and 10 µm, respectively. In addition, the final concentrations of GST-AD and untagged Ecotin-when they were included in the sample-were 4 µm and 40 µm, respectively. Following the oxidation reactions, the samples were boiled at 100 o C for 5 minutes. Next, one tenth of the samples were loaded onto a 10% Tricine-SDS polyacrylamide gel and separated by electrophoresis. The gel was then transferred onto a nitrocellulose membrane, probed with Avidin-HRP (Pierce, 5 µg/ml), and visualized by chemiluminescence (Pierce, SuperSignal West Pico Chemiluminescent Substrate) To affinity label the GGH-gpIII proteins on the fd-tet phage, 5 X 10 11 phage particles were incubated with 100 µm of Ni(OAc) 2 for 2
15 minutes at room temperature. Next, 250 µm of Biotin-tyramine was added to the solution. This was followed by the addition of 100 µm of MMPP to a final reaction volume of 200 µl. After 2 minutes of incubation at room temperature, the reaction was quenched with 75 µl of loading buffer that contained 6M of urea. Finally, 65 µl of the sample was loaded onto an SDS-PAGE gel for analysis. II- The quantification of affinity labeling To estimate the moles of biotin-tyramine that transfer to 1 mole of GGH-Ecotin, the chemiluminescence volumes given off by 1 mole of biotin and 3 label transfer reactions were measured. The measurements were then used in a series of calculations to estimate the moles of biotin-tyramine that transfer to 1 mole of GGH-Ecotin. Step I: The measurement of the chemiluminescence volume of 1 mole of biotin GGH-Ecotin was biotinylated by Sulfo-NHS biotin (Pierce product #21430). Different amounts were then separated by SDS-PAGE, probed with APS-Avidin (Pierce product #31002) and subsequently reacted with Amersham s AttoPhos signal amplification reagent (Product # RPN 5750). The chemiluminescence volume of each reaction was then measured by Amersham s STORM fluorimager (Data 3
Set A, Column 2). Subsequently, the chemiluminescence volume of each reaction was divided by the moles of Ecotin in the reaction (Column 1) to calculate the chemiluminescence volume of 1 mole of biotinylated GGH-Ecotin (Column 3). To calculate the chemiluminescence volume given off by 1 mole of biotin, the number of moles of biotin per 1 mole of biotinylated GGH-Ecotin was measured by the Pierce HABA assay (Pierce product # 21430). Based on the assay, 1.7 moles of biotin were fused to 1 mole of biotinylated GGH-Ecotin. Hence, the chemiluminescence volumes of 1 mole of biotinylated GGH-Ecotin (Column 3, Data Set A) was divided by 1.7 to determine the chemiluminescence volume of 1 mole of biotin. The results are summarized in Data Set B. Step II: The measurement of the number of moles of biotin that transfer to one mole of Ecotin. Three label transfer reactions were performed, and their chemiluminescence volumes were measured (Data set C, column 2). The number of moles of biotin in each reaction was then calculated by dividing the reaction s chemiluminescence volume by the measured chemiluminescence volume of 1 mole of biotin from data step B. The calculations are summarized in column 3 of Data Set C. Finally, the number of moles of biotin transferred to 1 mole of GGH-Ecotin was calculated by dividing column 3 with the number of moles of GGH-Ecotin (column 1, Data Set C). The results are summarized in column 4 of Data Set C. 4
Data Sets Data set A The chemifluorescence volume of 1 mole of biotinylated ecotin Bitinylated Ecotin (Moles) Chemifluorescence Volume Chemifluorescence Volume of 1 mole of biotinylated ecotin 1.37E-12 1245771 9.09E+17 2.74E-12 1850428 6.75E+17 5.49E-12 4123866 7.51E+17 Average Standard deviation 7.79E+17 1.19E+17 Data set B The chemifluorescence volume of 1 mole of biotin Moles of biotin per 1 mole of Ecotin (HABA) Chemifluorescence volume of 1 mole of biotinylated Ecotin (data set A) Chemifluorescence volume of 1 mole of biotin Standard deviation 1.7 7.79E+17 4.58E+17 7.02E+16 Data set C The number of moles of biotin label transferred to 1 mole of Ecotin Moles Ecotin Chemifluorescence volume Moles biotin per reaction Moles biotin per mole Ecotin 7.32E-12 7.32E-12 7.32E-12 16829825 2.16E-11 2.95E+00 23280379 2.99E-11 4.08E+00 23178938 2.98E-11 4.07E+00 Average 3.70E+00 Standard 0.648 Deviation 5
III- The synthesis of biotin-tyramine Biotin (100 mg, 409 µmol), DCC (90 mg, 436 µmol), and NHS (55 mg, 478 µmol) were dissolved in 10 ml of N-N -dimethylformamide (DMF) and stirred overnight at room temperature under N 2 pressure. A white precipitate of DCU was observed the next day and filtered. Next, tyramine (168 mg, 1.2 mmol) was dissolved in DMF and the solution was stirred at room temperature for two hours. A white precipitate of NHS was observed and filtered. The solvent was then removed and the product was re-dissolved in 500 µl of 60% CH 2 Cl 2, 20% MeOH, and 20% CHCl 3. Next, the product was purified from excess tyramine by silica gel chromatography in the same solvent that it was re-dissolved in. The fractions were then analyzed by TLC and visualized in an I 2 chamber. The R f s of tyramine and biotin-tyramine were 0.08 and 0.75, respectively. The fractions that only contained biotin-tyramine were pooled and their solvent was removed by drying under vacuum pressure. The final amount of isolated product was 16 mg, which corresponded to a 10% yield. The product was analyzed by mass spectrometry (Electrospray-MS m/z 364.08) and 1H NMR (300 MHZ, [D 6 ] DMSO, 25 o C, TMS). The product was then dissolved in DMSO to a final concentration of 250 mm, and stored at 20 o. All the starting materials and solvents were purchased from Sigma (St. Louis, MS). 6
Abbreviations (in the order used): DCC (dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide), DCU (dicyclohexylurea), CH 2 Cl 2 (dichloromethane), MeOH (methanol), CHCl 3 (chloroform), TLC (thin layer chromatography), R f (retention factor), and DMSO (dimethyl-sulfoxide). 7