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, FmocGly-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
(Marktredwitz,
Germany).
N-methylpiperidine,
Iris Biotech GmbH
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- Peptides 1 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 0.1 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.
Figure S1. Occurrence of amino acids in the prepared peptides
Amino acids relative
abundance (%)
* Swiss-Prot
40
35
Natural peptides *
Synthetic peptides
30
25
20
15
10
5
0
Aliphatic
L, A, G, V, I
Basic
K, R, H
Acid
Not charged
Aromatic
E, D
S, P, T, M, C, N, Q
W, F, Y
Amino acids
Intens. [a.u.]
Figure S2. MALDI MS/MS spectra of peptide 13 obtained upon a) LID/CID and b) LID
conditions
[M+H]+ 1544.74
S2a) peptide 13 (KWFGMLADQATYN)
2500
2000
1500
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
b-NH3
a-NH3
b8
949.33
y-NH3
Immonium Ion-NH3
Internal Ion-NH3
b6
763.29
b7
834.27
1000
b9
iK*
GMLADQA
84.03
1077.36
b12
b10
1412.73
1148.42
b5
500
iK
GML
TY
iW
159.05
WFG
1249.48
735.29
FGM FGML
FGMLAD
ADQ
b3
FGMLA
315.07
b1
b11
a6
650.20
a7
520.14
687.20
449.12
806.32
a10
574.20
391.10
b13
a12
1384.65
1120.54
1478.33
265.07
iMiY
0
200
400
600
800
1000
1200
1400
Intens. [a.u.]
m/z
[M+H]+ 1544.755
S2b) peptide 13 (KWFGMLADQATYN)
3000
b8
949.229
2000
b7
b6
iK*
834.200
763.216
84.252
b9
GMLADQA
b10
1077.314
b12
1148.337
b11
1412.616
1249.467
1000
GMLA
FGM
b1
ADQ
129.304
GM DQ
189.202 244.149
315.130
TY
b5
ADQA
FGMLAD
650.161
a6
WFG
LADQ LADQA574.134
428.098
735.213
687.105
b13
a10
a7
806.261
1460.179
499.105
373.116
0
200
400
600
800
1000
1200
1400
m/z
Intens. [a.u.]
Figure S3. LID/CID MALDI MS/MS spectrum of arginine-containing peptide 10
S3. peptide 10 (KYIWLSRAV)
8000
6000
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
Scrambling*
b-NH3
1036.643
a-NH3
y-NH3
Immonium Ion-NH3
Internal Ion-NH3
[M+H]+
1135.658
iK*
84.066
4000
b7
947.551
iW
iL
159.089
2000
b1
iK
129.101
IW/WL
b2 300.189
y3
IW/WL-CO
272.184
197.161
iY
LS
a2
345.256
FGM
b3
405.330
y4
y5
432.336
a3
377.312
b4
591.419
545.428
486.288 526.375
a4
-59
b5
704.483
a5
a8
y6
990.631
731.468
b6
676.518
463.302
y6*
518.425
y7
844.497
799.453
b7*
919.599
a7
1076.636
b8
b8*
-44
-42
0
200
400
600
800
1000
m/z
Intens. [a.u.]
Figure S4. LID/CID MALDI MS/MS spectra of proline-containing peptides a) peptide 6
(KYPFEAL), b) peptide 9 (KEDFPQLMV).
x104
S4a) peptide 6 (KYPFEAL)
2.0
1.5
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
576.28
y5
b-NH3
a-NH3
y-NH3
Immonium Ion-NH3
Internal Ion-NH3
1.0
y6
739.33
PFE
[M+H]+
374.18
PFEA
867.39
445.21
b5
0.5
YP
iK*
iP
84.06
FE
245.12
iK b1
101.09
PF
-CO
iY
y2
129.08
155.05
b2
217.11
203.08
b6
665.27
261.10
292.17
FEA
y3
332.18
348.16
PFEAL
b4
y4
a4
b3
468.50
389.22
YFP
536.28
YPFEA
a5
637.29
508.26
558.28
-46
a6
708.33
608.25
754.34
b6*
821.42
0.0
100
200
300
400
500
600
700
800
Intens. [a.u.]
m/z
S4b) peptide 9 (KEDFPQLMV)
y5
587.29
4000
3000
b7
858.37
b8
2000
989.43
[M+H]+
1106.57
b4
PQL
PQ
1000
b6
520.21
745.30
339.15
PQLM
226.07
DFPQL
470.21
iK*
b3°
a4
84.06
iP iK
101.07
b3
198.05
311.13
830.41
b5
492.21
y5*
373.15
iF
120.04
a6
y6
617.23
a6
392.09
964.26
0
200
400
600
800
1000
m/z
Intens. [a.u.]
Figure S5. MS/MS spectra of peptide 15 (KTRYNGMGEQWDPD) a) LID/CID in MALDITof/Tof, b) CID in ESI-QqTof from (M+H)+ precursor ion, c) CID in ESI-QqTof from
(M+2H)2+ precursor ion.
x104
S5a) peptide 15 (KTRYNGMGEQWDPD)
1.0
0.8
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
b12
1466.57
b-NH3
a-NH3
y-NH3
Immonium Ion-NH3
Internal Ion-NH3
0.6
[M+H]+
1697.81
0.4
243.03
0.2
iR
iK*
83.97
iY
y2
iR
b7
b3
203.07
136.00
386.10
QW
315.04
b4
566.21
a4
a5
635.19
1581.70
1037.37
1369.60
1654.70
1323.44
b5
1080.44
1165.59
0.0
200
400
600
800
1000
1200
1400
1600
m/z
S5b) peptide 15 (KTRYNGMGEQWDPD)
b12
b3
GE
b9
b1
a9
iK
iR
a11
y2
b3*
iW
a5
b5
YGN
QWD a4
b12*
b9*
[M+H]+
S5c) peptide 15 (KTRYNGMGEQWDPD)
y2
b122+
b12
QWDP
b102+°
b102+*
QWD
b102+ b112+
QW/
-CO
b5
CEQ
b3
b92+
b4
y3
iK iW y2*
TRYNGMGEQWD
[M+2H]2+
b122+°
b122+*
RYNGMGEQWD
b9
[-NH3]
2+
b10
b7 b8
b9*
y10
a10
a11 b11
b12°
b12*
y13
Intens. [a.u.]
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.
b6/y6
S6a) peptide 7 (KMVNLHI)
6000
4000
723.36
b7
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
836.44
b-NH3
a-NH3
y-NH3
Immonium Ion-NH3
y3
Internal Ion-NH3
397.23
[M+H]+
982.54
2000
a6
b5
695.38
586.32
a7
y5
LH/HI
iH
iK*110.07
84.07
b1
LHI
MVN 365.20
LH/HI-CO
223.14
129.08
y2/b2
y7
a5
510.29
251.13
525.27
b4
808.47
624.33
y4
558.34
854.43
MVNLH
b4*
MVNLHI
456.25
345.16
436.25
0
100
200
300
400
500
600
700
800
900
1000
m/z
LH / HI
S6b) peptide 7 (KMVNLHIQ)
y3
b6
-CO
NLH
a6
iH
VNLHI
VNLH
y2/b2
NLHI
MVN
b1 y1
b7
a7
b5* b5
b4*
y4
iK*
b4
[M+H]+
a6*
a5
a7*
VNL
VN
b6*
MVNLH
y5
b7*
-NH3
S6c) peptide 7 (KMVNLHIQ)
b1
[M+2H]2+
iK*
y1
b62+
y2/b2
iK
iH
y3
a62+
LH/HI
iL
MVN
iM
MV-CO
-CO
NLH
a62+*
a3
VNR
b72+
VNLH [-NH ]2+
3
-CO
VNLH
y72+
MVNL
y4
VNLHI
MVNLH
b5 y5
y6/b6
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.
S7a) peptide FVAEKFA
b6
y3
b5
b4
b1
b
b*
N terminal Ion
b°
b-H2O
a
a*
b-CO
a°
a-H2O
y
y*
C terminal Ion
y°
y-H2O
iX
iX*
Immonium Ion
iX°
Immonium Ion-H2O
XX
XX*
Internal Ion
XX°
Internal Ion-H2O
b-NH3
a-NH3
y-NH3
Immonium Ion-NH3
Internal Ion-NH3
y5
y4
AEK
b2
EK
b3
b2°
b6°
y4°
iK*
iF
iE
a6
a4
AE
a2
[M+H]+
y6
VAE
a5
S7b) peptide AFAMVGKLAE
b8/y8
y5
b9
b4
b3
y6
y4
b7
y7
VGK
a8°
GK
b1
VGKL
b2/y2
iK*
a4
a5
b6
AMVG
KLAL
a8*
y9
a9
[M+H]+
S7c) peptide WGVYAPLFDK
y5
y6
y7
y3
PL
iW
iP
b2
y2
PLFD
PLFGK
b3
b3
b5
a3
YAPL
y8
b7
a5
PLF
a6 b6
a7
b8
a8
y9
[M+H]+