Supporting Information for: Oncocin (VDKPPYLPRPRPPRRIYNR-NH 2 ): a novel antibacterial peptide optimized against Gram-negative human pathogens Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler, Annegret Binas, Oliver Nolte, Lisandra L. Martin and Ralf Hoffmann Contents: Table S1 lists all reagents used in this study including their purities and the suppliers. Table S2 lists the media used to culture the bacteria and eukaryotic cells Page S2: Statement on the purity of the synthesized peptides Figures S1 to S12: Analytical reversed-phase HPLC chromatograms and MALDI mass spectra for purified peptides 1 to 12. S1
Table S1: Reagents used in this study including their purities and the suppliers. Reagent Purity Supplier Fmoc-amino acid derivatives >99% (HPLC) Orpegen Pharma, Heidelberg, Germany 5,6-Carboxyfluorescein For fluorescence >95% (HPLC) Fluka Chemie GmbH; Buchs (Switzerland) 5,6-Carboxytetramethylrhodamine For fluorescence Merck, Darmstadt (Germany) >85% HPCE Acetonitrile HPLC-S, >99.95% (GC) Biosolve; Valkenswaard (Netherlands) Dimethyl sulfoxide Puriss., absolute over molecular Biosolve sieve, >99.5% (GC) Ethane dithiole Purum >98% (GC) Fluka Chemie GmbH Hydroxybenzotriazole Puriss >99% Fluka Chemie GmbH; m-cresole p.a. >99% (GC) Fluka Chemie GmbH; N,N'-Diisopropylcarbodiimide Purum >98% (GC) Fluka Chemie GmbH N,N- Peptide synthesis grade Biosolve Dimethylformamide >99.8% (GC) Sodium chloride For microbiology SIGMA-Aldrich-Laborchemikalien; Taufkirchen (Germany) Thioanisole Purum 99% (GC) Fluka Chemie GmbH Trans-retinoic acid >95% by assay Calbiochem, Darmstadt (Germany) Trifluoroacetic acid for Purum >98% (T) Fluka Chemie GmbH peptide synthesis Trifluoroacetic acid for Spectroscopy grade >99% Fluka Chemie GmbH HPLC Diethyl ether >99% (GC) Biosolve Sodium hydroxide >99% Carl Roth GmbH & Co. KG S2
Table S2: Media used in this study to culture the bacteria and eukaryotic cells and the suppliers Medium Purity Composition Supplier Nutrient solution for bacteria For microbiology Meat extract (3 g/l) Peptone from gelatine (5 g/l) Carl Roth GmbH & Co. KG; ph 6.8 ±.2 Tryptic soy broth For microbiology Casein peptone (pancreatic, 17 g/l) Dipotassium phosphate (2.5 g/l) Glucose (2.5 g/l) Sodium chloride (5 g/l) Soy peptone (3 g/l) ph 7.3 ±.2 Fluka Chemie GmbH Statement about the peptide purity: General procedures for all peptide syntheses, purifications, and analyses were performed as described previously (Singer, D.; Lehmann, J.; Hanisch, K.; Hartig, W.; Hoffmann, R. Biochemical and Biophysical Research Communications 6, 346, 819-828.). The purified peptides were analyzed by RP-HPLC on a Jupiter C 18 -column (4.6 mm internal diameter, 15 mm length, 5 µm particle size, 3 nm pore size; Phenomenex) using a linear gradient from 5 to 33.6% aqueous acetonitrile for 17 min in the presence of trifluoroacetic acid as ion pair reagent. The purities were above 95% based on the peak areas obtained after background correction. The molecular weight of the peptides were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; 47 proteomic analyzer; Applied Biosystems GmbH, Darmstadt, Germany) using α-cyano-4-hydroxycinnamic acid (4 mg/ml in 6% aqueous acetonitrile containing.1% TFA) as matrix (see Supporting information). S3
Figure S1: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 1. 1 A25 A4 C6 (1274974944)2:1_UV2_214nm 17.31. 5. 1. 15. 2. 25. min 9 8 7 2445.4141 2.5E+4 6 5 4 3 2 1 55.1544 656.84 877.54 1223.7129 213.1821 1 19 2 3 2289.2847 2428.8359 2546.4978 S4
Figure S2: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 2. 2 A25 A5 G3:1_UV2_214nm 17.22. 5. 1. 15. 2. 25. min 9 8 7 2449.386 1.8E+4 6 5 4 3 2 1 568.157 666.294 877.539 1225.6943 217.1619 1 19 2 3 2293.263 2471.3647 S5
Figure S3: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 3. 3 A28 B2 G4:1_UV2_214nm 17.3. 5. 1. 15. 2. 25. min 9 8 7 2485.4158 1.5E+4 6 5 4 3 2 1 568.1582 666.32 877.54 1243.713 257.3918 1 19 2 3 S6
Figure S4: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 4. 4 A29 B1 C5:1_UV2_214nm 16.85. 5. 1. 15. 2. 25. min 9 8 7 2476.4417 1.4E+4 6 5 4 3 2 1 56.163 568.1541 666.37 877.537 184.6342 1239.2231 2134.2227 1 19 2 3 232.346 2498.4219 S7
Figure S5: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 5. 5 A25 A6 A16:1_UV2_214nm 17.1. 5. 1. 15. 2. 25. min 9 8 7 254.5725 726.4 6 5 4 3 2 1 656.96 1253.311 1 19 2 3 2234.4292 2348.4556 2488.81 S8
Figure S6: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 6. 6 A29 A2 D77:1_UV2_214nm 17.8. 5. 1. 15. 2. 25. min 9 8 7 253.4922 2.5E+4 6 5 4 3 2 1 65.62 1252.7439 1448.853 164.9491 2191.2671 1 19 2 3 2347.3616 2486.9292 2583.4399 S9
Figure S7: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 7. 7 A29 A6 B8:1_UV2_214nm 17.1. 5. 1. 15. 2. 25. min 9 8 7 254.4487 1.9E+4 6 5 4 3 2 1 568.164 666.327 877.538 1253.2 2162.2319 239.487 2487.8965 1 19 2 3 265.537 S1
Figure S8: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 8. 8 A35 A2 E9:1_UV2_214nm 16.96. 5. 1. 15. 2. 25. min 9 8 7 253.4133 2.7E+4 6 5 4 3 2 1 568.1527 666.492 877.541 1252.723 1351.7838 2161.1838 1 19 2 3 2347.2781 2486.8484 2648.5125 S11
Figure S9: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 9. 9 A35 A4 B91:1_UV2_214nm 17.13. 5. 1. 15. 2. 25. min 9 8 7 239.4241 6944.3 6 5 4 3 2 1 524.1732 656.867 877.543 1196.2174 1 19 2 3 2234.3 2412.3999 S12
Figure S1: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 1. 1 A66 B3 B5B611:1_UV2_214nm 17.5. 5. 1. 15. 2. 25. min 9 8 7 2389.3872 1.E+4 6 5 4 3 2 1 568.1481 666.286 877.536 1195.72 247.1776 2175.2791 1 19 2 3 2411.363 S13
Figure S11: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 11. 11 A53 F2 D112:1_UV2_214nm 16.71. 5. 1. 15. 2. 25. min 2913.5 9 8 235.3633 7 6 5 4 3 2 1 568.1524 524.1616 666.27 877.539 1153.697 291.2561 2288.3452 1 19 2 3 S14
Figure S12: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 12. 12 A51 A6 A1113:1_UV2_214nm 17.37. 5. 1. 15. 2. 25. min S15