SUPPORTING INFORMATION

Peptide synthesis:

We have used a 2-chlorotrityl chloride polystyrene resin or a trityl-alcohol ChemMatrix resin for the synthesis of C-terminal carboxylic acid peptides.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-Ile-OH, 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 Iris Biotech GmbH(Marktredwitz, Germany).HBTU [2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate] coupling reagent was purchased from Iris Biotech GmbH (Marktredwitz, Germany).N-methylpiperidine, N-methylpyrrolidone, 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),SIGMA-ALDRICH (St Quentin, 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 Iris Biotech GmbH (Marktredwitz, Germany) and Trityl alcohol-ChemMatrix resin was purchased from PCAS BioMatrix Inc. (Quebec, Canada).

Standard peptide Fmoc strategy was used for all peptides carried out on a 96 well Advanced Chemtech ACT496  multiple organic synthesizer (peptides 1 to 10) and on LibertyTM Microwave Peptide Synthesizer (peptides 11 to 15).

1-Peptides1 to 10 were synthesized on a 96 well Advanced Chemtech ACT496  multiple organic synthesizer.

The protocol has been published elsewhere [20].

2-The microwave synthesis of peptides 11 to 15 were performed by LibertyTM Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC), an additional module of DiscoverTM (CEM Corporation, Matthews, NC) that combines microwave energy at 2450 MHz to SPPS following the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy.

Syntheses were conducted on a 0.1 mmol scale. Deprotections were performed with a 20% piperidine in DMF solution. All coupling reactions were performed with 5 equivalents of HBTU in DMF (0.5 M), 5 equivalents of amino acids in DMF (0.2 M) and 10 equivalents of DIPEA in NMP solution (2 M).

Each deprotection and coupling reaction was performed with microwave energy and nitrogen bubbling. Microwave cycle was characterized by two deprotection steps; the first one was for 30 s, the second one for 180 s. All coupling reactions were for 300 s.

After the assembly was complete, the peptide-resin was washed with CH2Cl2.

Peptide cleavage from the resin and deprotection of the amino-acids side chains was performed with TFA/H2O/TIS solution (95:2.5:2.5 v/v/v). In all cases the cleavage was maintained for 3 h at room temperature. The resins were washed with TFA and the filtrates partially evaporated. The crude products were precipitated with diethyl ether, collected by centrifugation, dissolved in H2O/AcN and lyophilized.

Peptide purification:Samples were dissolved in an 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 X Bridge Prep C18 5 µM OBD 19x100 mm column. A flow rate of 20 mL/min and a gradient of 20–40% B over 5 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 nebulizing and drying gas. The data were obtained in a scan mode ranging from 100 to 1000 m/z in 0.1s 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.

Figure S1.Occurrence of amino acids in the prepared peptides

Figure S2.MALDI MS/MS spectra of peptide 13 obtained upon a) LID/CID and b) LID conditions

Figure S3. LID/CID MALDI MS/MS spectrum of arginine-containing peptide10

Figure S4.LID/CID MALDI MS/MS spectra of proline-containing peptides a) peptide 6 (KYPFEAL), b) peptide 9 (KEDFPQLMV).

Figure S5.MS/MS spectra of peptide 15 (KTRYNGMGEQWDPD)a) LID/CID in MALDI-Tof/Tof, b) CID in ESI-QqTof from (M+H)+ precursor ion, c) CID in ESI-QqTof from (M+2H)2+ precursor ion.

Figure S6. MS/MS spectra of peptide 7 (KMVNLHIQ) a) LID/CID in MALDI-Tof/Tof, b) CID in ESI-QqTof from (M+H)+ precursor ion, c) CID in ESI-QqTof from (M+2H) 2+ precursor ion.

Figure S7. LID/CID MALDI MS/MS spectra of lysine-containing peptides inside the sequence a) peptide FVAEKFA and b) peptide AFAMVGKLAE, and c) at the C-terminal position, peptide WGVYAPLFDK.