2. Protein expression and fluorescence labeling of the SH2
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1 Supporting information: 2 3 4 Sublibrary numbering and screening layout for the three phosphopeptide libraries 5 6 7 8 9 From each sublibrary of the pentapeptide libraries (yellow, blue) which contain a theoretical 3375 different peptides each, two wells of a 96-well microtiter plate (corresponding to ~ 4 000 beads) were filled with beads. For the tetrapeptide library (red) which contains only 225 peptides only one well was used. This resulted in a screening plate of 75 wells. The sublibrary allocation within a 96-well plate is shown in Table S1 and Figure S1 10 Table S1 Sublibrary numbering and R4 residues for library 3 Sublibrary R4 amino acid Well No. of peptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Ala Ile Val Phe Tyr Trp Pro Arg Lys Ser Thr Asp Glu Asn Gln Ala Ile Val Phe Tyr Trp Pro Arg Lys Ser Thr Asp Glu Asn Gln Ala Ile Val Phe Tyr Trp Pro Arg Lys Ser Thr Asp Glu Asn A1, B1 A2, B2 A3, B3 A4, B4 A5, B5 A6, B6 A7, B7 A8, B8 A9, B9 A10, B10 A11, B11 A12, B12 C12, D12 C11, D11 C10, D10 C9, D9 C8, D8 C7, D7 C6, D6 C5, D5 C4, D4 C3, D3 C2, D2 C1, D1 E1, F1 E2, F2 E3, F3 E4, F4 E5, F5 E6, F6 E7 E8 E9 E10 E11 E12 F7 F8 F9 F10 F11 F12 G12 G11 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 3375 225 225 225 225 225 225 225 225 225 225 225 225 225 225 45 Gln G10 225 1 2 3 4 5 6 7 8 9 10 11 For technical reasons each on-bead screening library used in the CONA screening process is composed of individual sublibraries. The screening of defined subsets and the picking from these subsets facilitates the decoding of hit structures. Each sublibrary is defined by a specific branch which it belongs to (yellow, blue and red) and by the identity of the last building block. E.g., ibrary 3 consists of 45 sublibraries, 15 sublibraries per branch.. For on-bead screeningthe sublibraries of the phosphopeptide library were numbered as listed above (1-15 :yellow branch, 16-30 :blue branch, 3145 :red branch). The sublibrary is denoted in the second column. For each screen, two wells of a 96 well plate were filled with beads from each sublibrary from the two first branches (yellow and blue) and one well only was filled with beads of the red branch. The well number(s) where the specific sublibrary is found within the screening plate are listed in the third column. The fourth column contains the number of compounds per sublibrary. 12 Figure S1 Sublibrary allocation within a screening plate 1 2 3 4 5 A 1 2 3 4 B 1 2 3 4 5 6 6 7 7 8 8 9 9 10 11 12 10 11 12 5 6 7 8 9 10 11 12 C 24 23 22 21 20 19 18 17 16 15 14 13 D 24 23 22 21 20 19 18 17 16 15 14 13 E 25 26 27 28 29 30 31 32 33 34 35 36 F 25 26 27 28 29 30 37 38 39 40 41 42 G 43 44 45 H 13 14 15 16 17 18 19 Screening plates, used for the phosphotyrosine library, consist of 75 wells. The numbers correspond to the sublibrary numbers from Table S1. The color coding indicates the affiliation of individual sublibraries to the three different branches for the library. Table S2 Number of hit beads per well and maximal ring intensities for Cy5-SAP Well No. Well Sublibrary No. of hits Maximum ring intensity [kHz] 1 A1 Ala 2 150 2 A2 Ile 0 3 A3 Val 4 150 4 A4 Phe 1 150 5 A5 Tyr 1 200 6 A6 Trp 0 7 A7 Pro 3 150 Well No. Well Sublibrary No. of hits Maximum ring intensity [kHz] 8 A8 Arg 25 400 9 A9 Lys 9 250 10 A10 Ser 0 11 A11 Thr 0 12 A12 Asp 0 13 B12 Asp 0 14 B11 Thr 0 15 B10 Ser 0 16 B9 Lys 5 200 17 B8 Arg 25 600 18 B7 Pro 2 200 19 B6 Trp 0 20 B5 Tyr 0 21 B4 Phe 3 150 22 B3 Val 3 250 23 B2 Ile 0 24 B1 Ala 4 200 25 C1 Lys 14 400 26 C2 Arg 12 500 27 C3 Pro 6 200 28 C4 Trp 5 800 29 C5 Tyr 2 800 30 C6 Phe 3 800 31 C7 Val 4 250 32 C8 Ile 5 400 33 C9 Ala 0 34 C10 Gln 0 35 C11 Asn 1 36 C12 Glu 0 37 D12 Glu 2 38 D11 Asn 0 200 500 Maximum ring intensity [kHz] Well No. Well Sublibrary No. of hits 39 D10 Gln 0 40 D9 Ala 1 200 41 D8 Ile 7 500 42 D7 Val 6 300 43 D6 Phe 6 700 44 D5 Tyr 4 400 45 D4 Trp 8 400 46 D3 Pro 7 500 47 D2 Arg 20 1000 48 D1 Lys 10 1000 49 E1 Ser 0 50 E2 Thr 3 51 E3 Asp 0 52 E4 Glu 0 53 E5 Asn 2 200 54 E6 Gln 4 300 55 E7 Ala 0 56 E8 Ile 0 57 E9 Val 1 300 58 E10 Phe 5 400 59 E11 Tyr 0 60 E12 Trp 3 61 F12 Asp 0 62 F11 Thr 0 63 F10 Ser 0 64 F9 Lys 0 65 F8 Arg 10 66 F7 Pro 0 67 F6 Gln 3 250 68 F5 Asn 5 400 69 F4 Glu 0 200 200 300 Maximum ring intensity [kHz] Well No. Well Sublibrary No. of hits 70 F3 Asp 0 71 F2 Thr 5 500 72 F1 Ser 2 200 73 G1 Glu 0 74 G2 Asn 0 75 G3 Gln 0 Beads in total 150 000 No. of hits: 253 hit rate: 0.0017 1 2 3 4 All beads with fluorescent ring intensities above 200 kHz were counted as hits. The number of hit beads is listed for each well of the screening plate. 5 6 Figure S3: Picked SAP hit-beads and their respective fl. ring intensities (RI) Pick label RI Pick label RI Pick label RI A8-021 234 C2-186 357 C1-060 359 A8-026 282 C2-298 426 C1-082 395 A8-048 420 C2-293 185 C1-092 406 A8-091 536 C5-027 461 C1-144 105 A8-109 224 C5-054 417 C1-148 124 A8-124 302 C5-279 147 C1-159 204 A8-209 394 C4-036 268 C1-167 138 A8-282 351 C4-236 154 C2-048 316 A8-357 312 D1-027 405 D6-392 236 A8-415 337 D1-028 1451 D6-395 477 A8-517 335 D1-094 515 D6-413 506 A8-210 331 D2-069 594 D8-057 441 B8-517 1003 D2-177 500 D8-237 325 C1-047 386 D3-355 494 D8-401 404 C1-034 420 D5-062 446 E10-078 225 C1-052 555 D6-277 486 E10-588 383 1 Figure S2 2D-FIDA anisotropy data for SAP hit compounds Off-bead confirmation SAP - part 1 Cpd only Fluorescence Anisotropy 0.1 SAP (16 µM) 0.08 SAP (16 µM) 0.06 0.04 C1-47 C1-12 B8-517 A8-210 A8-517 A8-415 A8-357 A8-282 A8-209 A8-142 A8-124 A8-109 A8-095 A8-048 A8-026 A8-021 pos c TMR 0.02 Bead # Off-bead confirmation SAP - part 2 Cpd only Fluorescence Anisotropy 0.1 SAP (16 µM) 0.08 SAP (16 µM) 0.06 0.04 C5-054 C4-236 C4-036 C5-27 C2-293 C2-298 C2-186 C2-048 C1-167 C1-159 C1-148 C1-144 C1-092 C1-082 C1-060 C1-34 C1-052 pos c TMR 0.02 Bead # Off-bead confirmation SAP - part 3 Cpd only Fluorescence Anisotropy 0.1 SAP (16 µM) 0.08 SAP (16 µM) 0.06 0.04 2 3 4 5 6 7 8 9 E10-588 E10-078 D8-401 D8-237 D8-057 D6-413 D6-395 D6-392 D6-277 D5-062 D3-355 D2-177 D2-069 D1-094 D1-028 D1-027 pos c C5-279 TMR 0.02 Bead # Single-point 2D-FIDA anisotropy solution measurements () were performed with samples derived from all picked hit beads from the Cy5-SAP screen. The cleaved compound fractions were diluted 1:500, incubated with SAP in a 384-well microtiter plate and measured on the PS02 (assay volume 7 µl, 12x12 sec measurement time). A literature described α-peptidic SAP binder, used in HPLC-purified form, served as positive control (denoted pos c). 1 Kd determination and quenching analysis 2 3 In the simplest case of a ligand - receptor interaction with a 1:1 stoichiometry the dissociation constant Kd is defined as R L RL 4 Kd 5 [R], [L]: free receptor and ligand concentrations; [RL]: complex concentration at equilibrium. 6 7 For a (PS/PS-labeled) sample containing the labeled ligand and a percentage of (non-binding competent) impurities fi, the mass balances are R L 8 9 10 11 [ES-2] L0 RL fi L0 [ES-3] and the fraction of bound ligand a, derived from the algebraic solution for the binding equation, is a b (b) 2 4( R0 L0 R0 f im L0 ) 2 L0 [ES-4] with b ( L0 R0 K d f im L0 ) 14 15 16 17 R0 RL R0, L0: total receptor and ligand concentrations. 12 13 [ES-1] [ES-5] <<why the use of minuses here, compared to the text…>>Any measured average steady state readout parameter y (i.e. anisotropy or translational diffusion time) is related to the degree of complex formation by y (min (max Q min) a) 1 (1 Q) a [ES-6] 20 Q qbound q free [ES-7] 21 22 23 24 25 min, max: starting and end values of the average steady state parameter; Q: quenching factor; qbound, qfree: molecular brightnesses of the labeled ligand in the bound and free state. For FIDA measurements, molecular brightnesses are obtained for each polarization channel and the molecular brightness values q can be calculated directly by q = q|| + 2x q. For FCS measurements, a possible quenching factor can be derived from the total intensities. 18 19 with 26 27 28 29 30 31 Equilibrium dissociation constants (Kd values) were obtained by performing a nonlinear least square regression fit of the fluctuation data sets, based on equation [ES-4 and ES-6] with the software package GraFit 5.0. Equation [ES-6] contains two fit parameters: the end-value max and the dissociation constant Kd. 32 Decoding and re-synthesis of SAP-hits 33 34 35 36 Table S4 summarizes the off-bead confirmation and decoding results from the Cy5-SAP screen. All six samples, for which a Kd in solution had been obtained, were analyzed by MALDI-MS and MS/MS measurements. Peaks in the range of the diagnostic Y-fragments (m/z: 856 to 2 000 Da) were compared to the theoretical fragmentation pattern. 1 2 3 With the exception of compound C4-236, all samples proved to be decodable and corresponding candidate structures were found. Compound C4-236 produced an MS-spectrum of very low intensity, which wasnot be interpretable. 4 Table S4 Decoding table for Cy5-SAP Sample # Name Branch 1 D3-355 Blue 2 C5-054 3 D6-392 Blue Blue Sub Kd (µM) Label/spacer R1 R2 aR3 pY R4 Ac Pro PEG-TMR b3F b3S Isoleucine pY b3P Ac PEG-TMR b3S b3F Isoleucine pY b3P Ac PEG-TMR b3V b3V Phenylalanine pY b3Y Ac PEG-TMR b3V b3F Valine pY b3Y Ac PEG-TMR b3D b3F Tryptophan pY b3F Ac PEG-TMR b3F b3D Tryptophan pY b3F Ac PEG-TMR b3V b3Y Tryptophan pY b3F Ac PEG-TMR b3Y b3V Tryptophan pY b3F Ac Tyr Phe 11 8 35 4 C5-279 Blue Tyr 70 PEG-TMR b3R b3R Tyrosine pY b3Y Ac 5 A8-124 Blue Arg 100 PEG-TMR b3W b3Y Tyrosine pY b3R Ac PEG-TMR b3Y b3Y Tyrosine pY b3R Ac PEG-TMR b3Y b3W Tyrosine pY b3R Ac 6 C4-236 Blue Trp 120 no interpretable MS-spectrum was obtained 5 6 7 8 List of candidate structures for titrated hits from the Cy5-SAP screen. The fragment composition is drawn from the C- to the N-amino acids are denoted in one-letter code as b3-amino -Phosphotyrosine and Ac stands for N-acetyl. 9 10 11 12 The MS-decoding resulted in a total of 12 candidate structures. Due to the low affinity of compound A8-124 for SAP, no follow up experiments were carried out with this compound. All other nine candidate structures were re-synthesized on TentaGel beads with the identical chemical setup as used for the screening library. 13 Table S5 Cpd NVP-VAY076 NVP-VAY079 NVP-VAY080 NVP-VAY081 NVP-VAY082 NVP-VAY083 NVP-VAY084 NVP-VAY093 NVP-VAY094 14 List of re-synthesized hit compounds from Cy5-SAP Name SAP-01 SAP-02 SAP-03 SAP-04 SAP-05 SAP-06 SAP-07 SAP-08 SAP-09 Label/spacer PEG-TMR PEG-TMR PEG-TMR PEG-TMR PEG-TMR PEG-TMR PEG-TMR PEG-TMR PEG-TMR R1 b3F b3F b3V b3V b3V b3S b3Y b3D b3R R2 b3D b3S b3F b3Y b3V b3F b3V b3F b3R aR3 Tryptophan Isoleucine Valine Tryptophan Phenylalanine Isoleucine Tryptophan Tryptophan Tyrosine pY pY pY pY pY pY pY pY pY pY R4 b3F b3P b3Y b3F b3Y b3P b3F b3F b3Y Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac 15 16 17 List of re-synthesized structures from the Cy5-SAP screen. The peptide sequence is drawn from the C- to the N-terminus. ß³-amino acids are denoted in one-letter code as b3-X, α-amino acids are written in full names, pY means α-Phosphotyrosine and Ac stands for N-acetyl. 18 Table S6 Scores obtained for hit series vs native SLAM peptide Ligand SAP-02 SAP-03 SAP-06 SAP-08 SAP-09 SLAM peptide unphosphorylated SLAM peptide phosphorylated Szybki Single Point Energy (kcal/mol) -6.82 1.40 -12.80 0.12 2.96 -41.98 -49.13 Binding Affinity (Kd) 17 ± 1 μM 31 ± 11 μM 13 ± 1 μM 30 ± 3 μM 68 ± 10 μM 300 nM 100 nM 1 2 3 4 5 6 7 Scores for native peptide (unphosphorylated and phosphorylated) were directly calculated from PDB complexes (1D4T, 1D4W). SAP-02, 03, 06, 08, 09 Scores were calculated from top ranked complexes generated within RosettaLigand prioritized by Interface Delta. Figure S3 Plot of Autoligand results indicating total energy per volume (kcal/molÅ3) versus volume (Å3). Total Energy per Volume [kcal/mol A**3] -0.12 -0.13 Fill_10 -0.14 Fill_25 -0.15 Fill_50 Fill_75 -0.16 Fill_100 -0.17 Fill_150 Fill_200 -0.18 Fill_300 -0.19 Fill_400 -0.2 Fill_500 Fill_600 -0.21 0 500 1000 1500 2000 Fill_700 Volume [A**3] 8 9 10 11 12 Figure S3. Plot of AutoLigand results of total energy per volume (kcal/molÅ3 ) versus volume (Å3 ). The legend indicates the number fill points used tofind find the optimal The legend indicates theof number of fill points used to the optimal fill volume. fill volume. Figure S4 Ligand and receptor-based pharmacophore generated with vROCS for SLAM binding pocket of SH2 domain. 13 14 15 16 17 1,2 indicate features that mapped co-crystallised waters of PDB entries:1D4T, 1D4W, which were subseqeuently removed from the pharmacophore. 1 2 3 4 Figure S5 Binding site residues and critical interactions observed between SLAM phosphopeptide and SAP SH2 binding domain. 5 6 Figure S5. Binding site residues and critical interactions observed between SLAM phosphopeptide and SAP SH2 binding domain. 7 Materials and Methods 8 1. General methods 9 HPLC-instrumentation 10 Analytical HPLC: 11 12 13 HPLC analysis was performed on an Agilent 1100 series HPLC system, consisting of a quaternary pump (G1311A), a degasser (G1322A), an FLD detector (G1321A) and a DAD detector; column: Vydac peptide C8, 4.6 mm x 150 mm, 5 µm particle diameter size. 14 15 16 Analyses were performed using a linear gradient of A: H2O containing 5% MeCN and 0.1% TFA and B: MeCN containing 5% H2O and 0.1% TFA with a flow rate of 0.8 ml/min with FAD detection at: absorption 555 nm and emission 575 nm. Retention times (tR) are denoted in minutes. 17 Standard gradient: from 5% to 45% ACN in 20 min, then to 95% in 5 min. 18 Preparativ HPLC: 19 20 21 22 23 24 Purification of mg quantities of peptides and dyes was carried out using a preparative HPLC system (Agilent 1100 prep-HPLC system), equipped with a preparative autosampler (G2260A), preparative scale pumps (G1361A), a fraction collector (G1364B-prep) and a multiwavelength UV detector (G13658 MWD with preparative flow cell). The crude material was dissolved in a DMF/water mixture (~ 2:1) and separated at a flow rate of 20 ml/min on an Agilent RP-C-18 column (21.2 x 150 mm, 10 µm particle size), using a water/acetonitrile gradient and a detection wavelength of 210 nm. 25 26 Solvent A: water, 5% acetonitrile, 0.1% TFA; solvent B: acetonitrile, 5% water, 0.1% TFA. Gradient: 5% to 40% B in 20 minutes, 40% B to 95 % B in 5 minutes. 1 ESI-MS instrumentation 2 3 The mass spectra were recorded on a Waters MS-70.4000 micromass spectrometer, fragment ions are given in m/z. 4 2. Protein expression and fluorescence labeling of the SH2-domain of SAP 5 6 7 8 The recombinant protein was cloned, expressed and purified as N-terminal thioredoxin (trx)-tag fusion construct. The purification and solubility tag was cleaved by PreScission Protease (Amersham Biosciences) in the last step of the purification, leaving a sequence of 4 residual amino acids GPGS and the 103 amino acids from the SH2-domain of SAP: 9 SAP-SH2 sequence: 10 11 GPGSDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYRVSQT ETGSWSAETAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEK 12 13 14 This sequence contains 107 aa (1 Trp, 9 Tyr, 7 Lys, 2 Cys), amounting to a molecular weight of 11854.5 Da. The theoretical extinction coefficient according to [ES-9] therefore = 19035 [l mol-1 cm-1]. 15 16 Reducing SDS polyacrylamide gel electrophoresis of the purified protein showed one single band in the expected mass range (Figure , lanes 2 and 3). 17 18 19 The recombinant protein was randomly labeled on lysines with the activated dye Cy5-Nhydroxysuccinimidyl ester (Amersham Biosciences, Cy5-Mono NHS ester: MW = 754.9) using a 15 mM NaHCO3 buffer at pH 8.4 and following the manufacturer`s protocol for Cy5 labeling. 20 21 22 23 24 25 26 The final concentration of labeled protein was determined by recording a UV absorption spectrum on an Agilent 8453 spectrophotometer in the single beam mode, using 50 µl UV-silica cells (Ultra-Micro Cuvettes, Agilent) and the sample buffer as reference solution. The buffer subtracted spectra were corrected for Raleigh scattered light according to Heirwegh, Meuwissen and Lontie. 1 This correction 4 assumes that the contribution of Raleigh scattering to the overand that any absorption of the protein solution in the range of 300 to 330 nm is exclusively due to scattering. Then the scattering corrected absorption is given by 27 A280 E280 2 E300 or 28 29 30 31 32 33 34 A280 E280 1.95 E330 [ES-8] with A280: protein absorption at 280 nm and E280, E300 and E330: measured absorption signal at the respective wavelength. The concentration of unlabeled protein was calculated according to the Bouguer-Lambert-Beer law using the corrected protein absorption at 280 nm and the molar protein extinction coefficient at 280 nm, 280. 280 was calculated by an empirical formula according to Gill and von Hippel2: 280 l M -1cm-1 5 500 # Trp 1 490 # Tyr 125 # Cystines [ES-9] 35 36 37 38 39 The protein concentration and average labeling ratio of Cy5-conjugated proteins was calculated from UV-VIS data of the purified Cy5-labeled protein and the free dye in the respective buffer. A correction factor for Cy5 dye absorption at 280 nm was determined from the spectrum of the free dye by forming the ratio of the absorption at 280 nm to the absorption maximum at 649-650 nm. 40 41 42 43 The protein concentration and the labeling ratio for the labeled protein were calculated from the absorbance value at the absorption maximum of Cy5-protein, 649 nm and the signal at 280 nm, corrected for contribution of Cy5 to the absorption at 280 nm. Additionally, straylight correction factors, based on the absorption at 330 nm were included. 1 2 3 Calculation basis: extinction coefficient of Cy5: ε = 250 000 [M-1cm-1]. A280 Cy5 - corrected E280 ( E649 x 0.0274) [ES-10] A330 Cy5 - corrected E330 ( E649 x 0.0434) [ES-11] A280 Cy5 & stray corr. E280 Cy5 corrected ( E330 Cy5 corrected x 1.929) [ES-12] 4 5 Protein labeling result: 6 Cy5-SAP 7 8 9 10 11 12 13 14 15 16 17 A total amount of 13.5 nmol SAPtr was used for buffer exchange to 0.15 M NaHCO3 buffer, pH 8.4. The stray-light corrected UV-quantification before labeling yielded 7.3 nmol protein, which were reacted with Cy5-NHS ester (3 equivalents). After a reaction time of 3 hours HPLC-analysis indicated a consumption of 56.8% of labeling reagent or 1.7 equ. of Cy5-NHS. After separation of unreacted dye by size exclusion the stock solution contained 3.16 nmol protein with a concentration of 5.27 µM and a protein/Cy5 labeling stoichiometry of 1/1.85. For CONA on-bead screening using the PS04 instrument, a minimal concentration of of protein of 1 nM dye equivalents are needed in order to obtain a high enough signal intensity for hit bead detection. Ideally, the concentration should be between 5 and 50 nM dye equivalents. Based in these specifications, the obtained Cy5 labeled SAP protein was found suitable for on-bead screening and used at a concentration of 5 nM (9.25 dye equivalents). 18 Figure S6 Absorption spectrum of unlabeled SAP Absorbance (AU) 1.35 1.15 0.95 0.75 0.55 0.35 0.15 -0.05 220 270 320 Wavelength (nm) 19 20 21 370 1 Figure S7 Absorption spectrum of Cy5-SAP Absorbance (AU) 0.95 0.75 0.55 0.35 0.15 -0.05 220 320 420 520 620 720 Wavelength (nm) 2 3 SDS-PAGE gel of Cy5-labeled SAP 0.5 µg SA P 0.5 µg So st Ma rk 12 0.2 0.2 97.4kDa 66.3kDa 55.4kDa 97.4kDa 66.3kDa 55.4kDa 97.4kDa 66.3kDa 55.4kDa 36.5kDa 31kDa 36.5kDa 31kDa 21.5kDa 21.5kDa 21.5kDa 14.5kDa 14.5kDa 14.5kDa 6kDa 6kDa 6kDa 3.5kDa 3.5kDa 3.5kDa 0.5 µg IR AK 4 0.5 µg MK 2 Ma r Figure S8 5µ gE RR α5µ Bi gE oti R n Ma Rγ rk -B iot 1 in Ma 2 rk 12 0.5 µg SA P 0.5 Ma µg rk So 1 s Ma 2 t 0.2 rk 5µ 12 gS 0 . 0.25µ AP5µg IR Ale gSA xa 48 APK4 8 0.5 -C µg y5 MK 2 Ma rk 12 4 36.5kDa 31kDa 97.4kDa 66.3kDa 55.4kDa 36.5kDa 31kDa 21.5kDa 14.5kDa 6kDa 3.5kDa 5 6 7 8 9 10 11 Reducing SDS-PAGE gel electrophoresis of unlabeled SAP-SH2 and Cy5-SAP-SH2. After labeling and purification by size exclusion chromatography the sample shows an identical purity as the unlabeled protein. For electrophoresis Novex pre-cast gels 4-12% Bis-Tris (Invitrogen) together with the Mark12 size standard (Invitrogen) were used. Protein bands were visualized, using the SILVER Express staining kit (Invitrogen). 12 Confocal Nanoscanning (CONA) hit bead ranking 13 14 15 16 17 Image analyses were carried out by the BeadEval software (Perkin Elmer, former Evotec). It first performs a bead-detection, based on predefined thresholds (e.g. bead diameter, ring width, minimal signal increase within a fluorescent ring, etc.). For each detected bead both, ring and area fluorescence intensities are then determined (Figure S9). 1 2 Figure S9 Schematic illustration of the parameters "ring intensity" and "area intensity" Bead Area Intnesity 1 – 2 µm Ring Intensity Scanning Laser Focus ~130 µm 3 4 5 6 7 8 9 10 Schematic illustration of the parameters “area intensity” and “ring intensity” used for evaluation of scanned beads. The area intensity is the average fluorescence intensity within the bead interior. The ring intensity is the average fluorescence intensity in the outer few µm of the bead area (= ring). The fluorescent ring is defined by a sharp and steep signal change in the outer regions of a bead. A recorded bead scan image corresponds to the optical cross-section through the bead along the scanning laser focus (dotted line). 11 12 13 14 15 16 17 For calculating the fluorescence ring intensity, the intensities of the brightest pixels in the neighborhood (in a ring of radius R + dr) of a detected bead are averaged. The area intensity of a bead is determined by averaging all pixel intensities within a circle of diameter 0.8 x R around the bead origin. To set a threshold for hit-bead classification the average level of background in the currently reported on-bead screen was determined and the threshold of hit-bead classification was set to four-times the average of background signal. 18 Chemical Syntheses 19 20 21 22 23 24 25 26 27 Abbreviations: Boc (tert-butoxycarbonyl), Boc2O (di-tert-butyl dicarbonate), DCM (dichloromethane), DIPEA (diisopropylethylamine), DMAP (4-(dimethylamino)pyridine), Fmoc (9fluorenylmethoxycarbonyl), Fmoc-OSu (9-fluorenylmethoxycarbonyl-N-succinimidyl-carbonate), HATU (O-(7-azabenzotriazol-1-yl) N,N,N,N - tetramethyluronium hexafluorophosphate, HMBA (4hydroxymethylbenzoic acid), HPLC (high performance liquid chromatography), h.v. (high vacuum (0.01-0.1 Torr), MeIm (1-methylimidazole), MSNT (1-mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4triazole), NMM (N-methylmorpholine), NMR (nuclear magnetic resonance), r.t. (room temperature), TFA (trifluoroacetic acid), TIS (triisopropylsilane), TMR (carboxy-tetramethylrhodamin), TNBS (2,4,6-trinitrobenzosulfonic acid), UV (ultra violet), eq. (equivalent) 28 29 30 Reagents: The Fmoc-protected a and 3-homoamino acids were purchased from Fluka, AnandChem GmbH or Chem-Impex International. The HMBA and Wang resins were purchased from Rapp Polymere. All other reagents were used as received from Sigma-Aldrich. 31 General procedures: 32 33 34 35 36 37 Anchoring of FmocPra-OH on HMBA resin (GP 1): Esterification of the Fmoc-protected propargylglycine amino acid with the HMBA resin was performed as follows: A solution of the Fmoc protected amino acid (5 equiv.) in dry DCM (3 ml/mmol) was treated with MeIm (3.75 equiv.) followed by MSNT (5 eq.) at r.t. After complete dissolution of the MSNT the solution was added to the pre-swollen resin (1 eq.). The suspension was mixed by Ar bubbling for 2h. Subsequently, the resin was filtered, washed with DCM (5 mL, 5 x 1min) and dried under h.v. for 24 h. 38 39 40 41 42 43 Solid-phase ß-peptide synthesis on the HMBA resin (GP 2): The Fmoc group of the first amino acid attached to the resin was removed using 20% piperidine in DMF (3 mL, 4 x 10 min). After filtration, the resin was washed with DMF (2 mL, 4 x 1 min). Solid phase peptide synthesis was then continued for each resin part by sequential incorporation of N-Fmoc-protected amino acids. For each coupling step, the resin was treated with a solution of N-Fmoc-protected amino acid (4 eq.), HATU (3.9 eq.), DIPEA (10 eq.) in DMF (1 mL) and mixed for 45-60 min. Monitoring of the coupling reaction was 1 2 3 performed with TNBS test. In case of a positive TNBS test (indicating incomplete coupling), the suspension was filtered off and the peptide resin was treated again with the same amino acid and coupling reagents. 4 5 6 7 8 9 10 N-terminal acetylation (GP3) After the last amino acid, the resin was Fmoc deprotected as above and the N-terminus was acetylated by treating the resin with a solution of Ac2O (10 equ.) and DIPEA (20 equ.) in DMF. After a reaction time of 1 h at r.t., the resin was drained and again treated with the same amount of fresh reaction mixture for another 1 h. Finally, the resin was washed extensively with DMF (4x3 min), DCM (4x3 min) and dried under h.v. for 12 h. 11 12 Side chain deprotection (GP4): 13 14 15 16 17 For side chain deprotection, each resin sample was treated with a solution containing TFA/H2O/Tis (95:2.5:2.5) at r.t.. After a reaction time of 2 h, water (10% v/v) was added to the reaction mixture and the reaction allowed to continue for further 16 h. The resin was then washed with DCM (4x2 min), DIPEA (10% v/v) in DCM (4x2 min), DCM (4x2 min) and dried in vacuo to yield the final resinbound peptide, which was stored at 4° C. 18 19 20 21 22 23 24 25 26 On-bead labeling and cleavage of batch synthesized peptides (GP 5): Several hundred beads are suspended in a mixture of 40 µL tBuOH and 35 µL H2O and treated with 20 µL of a freshly prepared solution of CuSO4 (3 mg) and ascorbic acid (1.5 mg) in 1 mL H2O. 30 µL of a solution of TMR-azide 5 in MeOH (approx. 5 mm) are then added and the reaction mixture is left to stand at r.t. for 24 h. The solution is then removed and the beads are washed 5-times with 100 µL MeOH and 5-times with 50 µL H2O. Finally, the labeled compound is cleaved from the bead by treatment of the resin with 120 µL of a cold (0° C) solution of NaOH (1n)-dioxane 1:3 for 15 min, followed by addition of 80 µL of HCl (1n). The solution is then purified by RP-HPLC. 27 28 Library Synthesis, general handling 29 30 31 32 All library syntheses were carried out by manual SPPS, using filter equipped, discard able syringes (2 ml, 5 ml and 10 ml from Becton Dickinson, NJ, USA) and fritted filter funnels for peptide synthesis (15 and 30 ml, Sigma-Aldrich) as reaction compartments and vacuum extraction devices (Malickrodt Baker, NJ, USA) for filtering and washing of resin samples within the syringes. 33 34 35 36 37 For the phosphopeptide library 1.5 g of TentaGel S HMB resin (loading: 0.26 mmol/g, custom sieved to achieve a size distribution of 90 to 100 µm diameter, Rapp polymers, Tübingen, Germany) were used. Before library synthesis each resin batch was checked for autofluorescence by placing 1 mg of swollen resin (PBS, 0.005% Tween20) in a 96-well microtiter plate and recording a CONA scan images. 38 39 40 41 42 43 44 45 46 After initial resin loading with Fmoc-propargylglycine according to GP1, the spacer unit, N-(Fmoc-8amino-3,6-dioxa-octyl)-succinamic acid, was coupled to the resin and the samples were subsequently subjected to Fmoc deprotection. For processing of combinatorial positions the deprotected resin samples were then split into sub-samples (one for each bulding block to be coupled), using Pasteur pipettes (Marienfeld laboratory glassware, Germany). To these N-terminally deprotected resin samples the respective amino acid building blocks were coupled according to GP2. Then the sub-samples were recombined in larger syringes and thoroughly mixed and washed. The mixed samples were again subjected to Fmoc-deprotection and re-split for the next coupling step. This split-mix-split steps were repeated according to the 1 2 3 library synthesis scheme (Fig. S5) until the last combinatorial coupling step, after which the resins were kept separated. (as sublibraries). Finally, the resin of each sub-library was N-acetylated (GP3) and subjected to side chain deprotection (GP4). 4 5 6 7 8 9 10 11 Single-bead PS/PS-labeling and library quality control For quality control, a random selection of beads were manually picked under a standard microscope using a syringe needle and placed - one bead per vial - into autosampler glass vials with a conical inlet (8002-SC-H, Glastechnik Gräfenroda, Germany). The PS/PS-labeling of the single beads cleavage of labeled material from solid support was carried out as described in the materials and method section in the main-text. The cleaved and dried material was dissolved in 20 µl of 20% acetonitrile (in water) and 15 µl were subjected to HPLC-analysis and compound quantification. 12 13 Resynthesized hit compounds from the phosphopeptide library pYL1 14 15 16 17 18 19 20 21 The MALDI-MS analysis and decoding yielded a series of hit-structures for each screen. These candidate structures were resynthesized on the TentaGel S HMB resin (loading: 0.26 mmol/g), using 20 mg resin per peptide (theoretical yield: 5.2 µmol peptide) and following the synthesis procedures described above. In brief: After resin loading with Fmoc-propargylglycine according to GP1, the N(Fmoc-8-amino-3,6-dioxa-octyl)-succinamic acid (ADO) spacer unit was coupled to the resin. After coupling of the spacer, the synthesis of the actual peptide sequences proceeded according to to the standard methods of building block coupling and Fmoc-deprotection GP2, N-Acetylation GP3 and side chain deprotection GP4. 22 23 Synthesis of unlabeled peptides: 24 Unlabeled hit compounds were synthesized without the spacer unit and the propargyl-glycine 25 26 27 28 29 30 31 32 33 34 labeling site, contained in the original hit. The synthesis was performed on a polystyrene PHB Wang resin (loading 1.15 mmol/g, Rapp polymere, Germany), typically using 50 – 100 mg of dry resin per peptide (theoretical yield: 58 - 115 µmol peptide). Loading of the first amino acid and peptide synthesis was performed according to GP1. Final side chain deprotection and resin cleavage was effected by treating the beads with a solution of TFA/H2O/Tis (95:2.5:2.5). After a reaction time of 2 hours, the resin was filtered off and washed 3 times with a small amount of cleavage solution. Water (10% v/v) was added to the combined filtrates and the solution agitated for another 16 hours. The solvents were then removed in vacuo and the crude peptide was precipitated by addition of cold ether, followed by repeated washing steps (3 times) with another aliquot of cold ether. The crude peptides were then purified by preparative RP-HPLC. 35 36 Detailed list of resynthesized peptides in PS/PS-labeled form 37 38 39 40 The compound identity was confirmed by ESI-MS, purity was assessed by RP-HPLC (fluorescence detection λex/λem 555/575nm. The purity values correspond to the crude PS/PS-labeled peptides, as this reflects best the actual purity on-bead. For solution testing, all peptides were HPLC-purified to yield purities > 90%. 41 1 Detailed list of re-synthesized hit-compounds in PS/PS-labeled form 2 3 4 The compound identity was confirmed by ESI-MS, purity was assessed by RP-HPLC (fluorescence detection ex/ em 555/575nm). The purity values correspond to the crude PS/PS-labeled peptides, as this reflects best the actual purity on-bead. 5 SAP-01 6 Sequence: 7 8 9 10 Ac-³hPhe-Tyr(PO3H2)-Trp-³hAsp-³hPheNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 17.12 min (74%). MS (m/z): [C90H105N15O22P+3H]3+ calc. 593.90, found: 594.2; [C90H105N15O22P+2H]2+ calc. 890.36, found: 890.2. 11 12 SAP-02 13 Sequence: 14 15 Ac-³hPro-Tyr(PO3H2)-Ile-³hSer-³hPhe-NH(CH2CH2O)2CH2CH2CONHCH2CH2COPra(TMR)-OH 16 17 RP-HPLC tR (%purity): 16.29 min (71%). MS (m/z): [C80H104N14O21P+3H]3+ calc. 543.57, found: 543.8; [C80H104N14O21P+2H]2+ calc. 814.86, found: 814.6. 18 19 SAP-03 20 Sequence: 21 22 23 24 Ac-³hTyr-Tyr(PO3H2)-Val-³hPhe-³hValNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 17.45 min (82%). MS (m/z): [C85H108N14O21P+3H]3+ calc. 564.92, found: 565.0; [C85H108N14O21P+2H]2+ calc. 846.88, found: 846.9. 25 26 SAP-04 27 Sequence: 28 29 30 31 Ac-³hPhe-Tyr(PO3H2)-Trp-³hTyr-³hValNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 20.29 min (46%). MS (m/z): [C91H109N15O21P+3H]3+ calc. 593.929, found: 594.1; [C91H109N15O21P+2H]2+ calc. 890.38, found: 890.3. 32 33 SAP-05 34 Sequence: 35 36 37 38 Ac-³hTyr-Tyr(PO3H2)-Phe-³hVal-³hValNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 19.48 min (76%). MS (m/z): [C85H108N14O21P+3H]3+ calc. 564.92, found: 565.1; [C85H108N14O21P+2H]2+ calc. 846.88, found: 846.6. 39 40 SAP-06 41 Sequence: 1 2 Ac-³hPro-Tyr(PO3H2)-Ile-³hPhe-³hSer-NH(CH2CH2O)2CH2CH2CONHCH2CH2COPra(TMR)-OH 3 4 RP-HPLC tR (%purity): 16.75 min (79%). MS (m/z): [C80H104N14O21P+3H]3+ calc. 543.57, found: 544.0; [C80H104N14O21P+2H]2+ calc 814.86, found: 814.7. 5 6 SAP-07 7 Sequence: 8 9 10 11 Ac-³hPhe-Tyr(PO3H2)-Trp-³hVal-³hTyrNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 19.61 min (65%). MS (m/z): [C91H109N15O21P+3H]3+ calc. 593.92, found: 594.1; [C91H109N15O21P+2H]2+ calc 890.38, found: 890.2. 12 13 SAP-08 14 Sequence: 15 16 17 18 Ac-³hPhe-Tyr(PO3H2)-Trp-³hPhe-³hAspNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 19.37 min (77%). MS (m/z): [C90H105N15O22P+3H]3+ calc. 593.91, found: 594.0. 19 20 SAP-09 21 Sequence: 22 23 24 25 Ac-³hPhe-Tyr(PO3H2)-Tyr-³hArg-³hArgNH(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH RP-HPLC tR (%purity): 14.35 min (64%). MS (m/z): [C87H114N20O22P+3H]3+ calc. 608.27, found: 608.6; [C87H114N20O22P+2H]2+ calc 911.91, found: 911.7. 26 27 Synthesis of a-phosphopeptides: 1.1.1 28 29 Ac-Thr-Ile-Tyr(PO3H2)-Ala-Gln-Ile-N(CH2CH2O)2CH2CH2CONHCH2CH2CO-Pra(TMR)-OH 30 31 RP-HPLC tR (%purity): 15.07 min (61%). MS (m/z): [C78H108N16O23P+2H]2+ calc. 556.91, found: 556.7 32 33 1.1.2 Synthesis of unlabeled hit-compounds for competition experiments 34 35 36 The compound identity was confirmed by ESI-MS, purity was assessed by RP-HPLC (absorption detection 220 nm). The purity values correspond to the purfied peptides, which were used for the competition experiments. 37 38 SAP-06 39 Sequence: 40 Ac-³hPro-Tyr(PO3H2)-Ile-³hPhe-³hSer-OH 1 2 RP-HPLC tR (%purity): 14.30 min (>95%). MS (m/z): [C37H51N5O12P+K]+ calc. 827.29, found: 827.6. 3 4 5 6 7 8 9 10 11 Reference List 12 13 14 15 16 17 18 19 1. Heirwegh, K. P. M.; Meuwissen, J. A. T. P.; Lontie, R., Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions. Journal of Biochemical and Biophysical Methods 1987, 14, (6), 303-322. 2. Gill, S. C.; von Hippel, P. H., Calculation of protein extinction coefficients from amino acid sequence data 1. Anal.Biochem. 1989, 182, (2), 319-326.
