Figure A: Peptide 4 (H-FPALVPI-NH2) analyzed at a concentration of 10-3M on a) porous silica gel and b) Pencil lead (2B) scratched on the MALDI target. Figure B: Peptide 4 (H-FPALVPI-NH2) analyzed at a concentration of 10-5M on a) porous silica gel and b) NALDITM-target. Peptide synthesis: As solid supports, we used a Rink amide polystyrene resin for the synthesis of C-terminal amidated peptide and a 2-chlorotrityl chloride resin for the synthesis of C-terminal carboxylic acid peptide. N- Fmoc protected amino acids (Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-IleOH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Asn(Trt)-OH) were purchased from SENN chemicals (Gentilly, France) and from Advanced Chemtech (Louisville, USA). HBTU [2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate] coupling reagent was purchased from SENN chemicals (Gentilly, France). N-methylpiperidine, N,N-dimethylformamide, dichloromethane, methanol, acetonitrile, diethyl ether, trifluoroacetic acid, N,N-diisopropylethylamine, piperidine and triisopropylsilane were purchased from Riedel-de Haën (New Jersey, USA), Carlo Erba (Val de Reuil, France) or Acros organics (Noisy le Grand, France) and used without purification. 2-Chlorotrityl chloride polystyrene resin (100-200 mesh, 1.48 mmol/g) was purchased from CBL/Patras (Patras, Greece) and Rink amide polystyrene resin (100-200 mesh, 0.98 mmol/g) was purchased from SENN chemicals (Gentilly, France). Standard peptide Fmoc strategy was used and peptides were synthesized on a 96 well Advanced Chemtech ACT496 multiple organic synthesiser. Each well was filled with 100 mg of resin. After anchoring of the first residue on the resin linker, deprotection and coupling steps were carried out until the desired sequences were synthesized. Anchoring: For C-terminal carboxylic acid peptide synthesis, freshly reinduced 2-chlorotrityl chloride resin (100 mg, 1.48 mmol/g) was suspended in 2 ml of anhydrous dichloromethane prior to the addition of SOCl2 (28 µl, 2.6 equivalents, 0.384 mmol). Resin was stirred for two hours. Resin was filtered and washed twice with anhydrous dichloromethane and used immediately for the first amino acid anchoring. 4.5 equivalents (0.666 mmol) of the first Fmoc-protected amino acid were dissolved in 2 ml of dichloromethane and were added to 100 mg of freshly reinduced chlorotrityl chloride resin. 233 µl of N,N-diisopropylethylamine (9 equivalents; 1.332 mmol) were then added. Resin was stirred overnight and washed as usual. For C-terminal amidaded peptides, 100 mg of Fmoc-Rink amide resin was conditioned for 10 minutes in dichloromethane and submitted to the standard deprotection cycle, using dimethylformamide / piperidine solution (80/20, v/v). After the washing step, the first amino acid was loaded onto the resin through a standard coupling cycle, using HBTU and Nmethylmorpholine. Coupling step: Three solutions were successively added to the reaction vessels: 400 µl of a 0.5 M solution of the N-Fmoc protected amino acid in N-methylpyrrolidone; 400 µl of a 0.5 M solution of N-methylmorpholine in dimethylformamide and 400 µl of a 0.5 M solution of HBTU in dimethylformamide. Coupling time was 90 min. Deprotection step: Fmoc deprotection was carried out using 1.2 ml of a dimethylformamide / piperidine solution (80/20, v/v) for 20 minutes. Washing steps: Washing steps were performed between coupling and deprotection steps. Resins were subjected to five consecutive washings using the following solvents: dimethylformamide (2 x 1.5 ml), dichloromethane (1.5 ml), methanol (1.5 ml) and dimethylformamide (1.5 ml). Cleavage: Peptides were cleaved from the resin for 2 hours in the robot’s 96-reactors block using 1.5 ml of cleavage cocktail (trifluoroacetic acid/water/triisopropylsilane, 95/2.5/2.5 v/v/v) per well. After removal of the resin by filtration, the cleavage cocktail was removed in a Jouan RC10-10 vacuum centrifugator. Compounds were precipitated three times by diethyl ether, the precipitate was dissolved in an acetonitrile/water (50/50, v/v) solution containing 0.1% trifluoroacetic acid and the solution was freeze dried. Peptide purification: Samples were dissolved in acetonitrile/water (50/50 v/v) mixture, containing 0.1% TFA. The LC/MS autopurification system consisted of a binary pump Waters 2525, an injector/fraction collector Waters 2676, coupled to a Waters Micromass ZQ spectrometer (electrospray ionization mode, ESI+). All purifications were carried out using a Chromolith SemiPrep RP-18 10x100 mm, monolithic column. A flow rate of 20 mL/min and a gradient of 0–20% B over 10 min was used. Eluent A: water/0.1% TFA; eluent B: acetonitrile/0.1% TFA. Positive ion electrospray mass spectra were acquired at a solvent flow rate of 204 L/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a scan mode ranging from 100 to 1000 m/z in 0.1 s intervals; 10 scans were summed up to get the final spectrum. Collection control trigger was set on single protonated and diprotonated ion with a MIT (minimum intensity threshold) of 7.105.