Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Peptide synthesis
Peptides were synthesized according to published methods [Fmoc Solid Phase Peptide
Synthesis, A Practical Approach, (W. C. Chan, P. D. White Eds), Oxford University Press,
2000. Solid Phase Synthesis, A Practical Guide, (S. F. Kates, F Albericio Eds), Marcel
Dekker, 2000] using standard solid-phase synthesis techniques with Fmoc chemistry, on a
433A peptide synthesizer (Life Technologies). Protected amino acids and chemicals were
purchased from Life Technologies, Novabiochem, Merck and Sigma. The resin loaded with
Fmoc-L-Val (A31-40) or Fmoc-L-Ala (A33-42) on the polyethyleneglycol/polystyrene
support (Fmoc-L-Val/ Ala-PEG-PS) with 4-hydroxymethylphenoxyacetic acid (HMPA)
linker was from Life Technologies. Fmoc-L-Val/ Ala-PEG-PS resin (0.25 mmol/ substitution
of 0.20meq per g) was treated with piperidine in NMP (N-Methyl-2-pyrrolidone) and linked
with Fmoc-L-Val (A31-40) or Fmoc-L-Ile (A33-42), with HBTU (2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)/ HOBt (N-Hydroxybenzotriazole) in
presence of DIEA (N,N-Diisopropylethylamine) as coupling reagents. All the other Fmoc
amino acids were sequentially coupled to the growing peptide chain and the coupling reaction
time was around 1h for all the natural and unnatural amino acids (X and a). A cysteine (FmocL-Cys (Trt) from Life Technologies) was introduced at the N-terminal of each peptide.
Piperidine was used to remove the Fmoc group at all steps. After deprotection of the last
Fmoc group, the peptide resins were washed with dimethylformamide, dichloromethane and a
dichloromethane/methanol solution (50/50_ v/v) and dried in vacuum to yield the protected
peptide HMPA-PEG-PS-Resin. The protected peptides-resins were treated with trifluoroacetic
acid/H2O/phenol/ethanedithiol/thioanisole (reagent K: 40 mL per 1 g of resin) for 2h. After
filtration of the exhausted resin, crude peptides were triturated with 250mL ether and purified
by high performance liquid chromatography using a C18 preparative reverse phase column
(C18 Delta Pack, 15µm, 300Å, 40x100mm from Waters). Conditions for the elution of
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
peptide C-KKK-PADRE-A31-40: the column was perfused at a flow rate of 35 mL/min with
a mobile phase containing buffer A (0.1% TFA in water) and buffer B (0.08% TFA in 80%
acetonitrile), using a multi-step elution gradient (10 to 39% buffer B for 15 min, 39 to 49%
buffer B for 10 min and 49% to 59% buffer B for 30min). The conditions for the peptide CKKKGS-A33-42 were the following: mobile phase containing buffer A (0.1% TFA in
water), buffer B (0.08% TFA in 60% acetonitrile) and the multi-step gradient: 20 to 41%
buffer B for 15 min, 41 to 51% buffer B for 10 min and 51 % to 61 % buffer B for 30 min.
The fractions containing the pure peptide were pooled and lyophilized. The peptides were
analyzed by high pressure liquid chromatography (HPLC) on a C18 reverse-phase column
(C18 Delta Pack, 5 µm, 300Å, 150x4.6 mm column from Macherey-Nagel), using a linear
elution gradient of 25 to 60 % buffer C (0.08% TFA in acetonitrile) and run for 30 min at a
flow rate of 1 ml/ min. The purity of the C-KKK-A33-42 and the C-KKK-PADRE-A31-40
peptides was estimated respectively at 75.0% and 94.0% by peak integration (peak with
absorption at 214 nm). The integrity of the peptides was also assessed by electrospray analysis
using a system mass spectrometer ESI-TOF (Life Technologies). Both peptides were coupled
via their N-terminal cysteine residue to maleimide-activated mcBSA (#77607 Pierce
Conjugation Kit, Rockford, USA) for immunization.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Generation of antibody-producing hybridoma clones
Ten micrograms of BSA-PADRE-A31-40 or BSA-A33-42 immunogens were emulsified in
equal ratio with Alum adjuvant (Thermo) and used to immunize either C57/Bl6 or CD1 mice
(Charles River), respectively, by intraperitoneal injection. Two booster injections (same dose)
were administered at 2-week intervals. Mice were bled 10 days after each boost and serum
titers were assessed by indirect ELISA to select the more responding mice. A pre-fusion boost
was administered 4 days before fusion. Mice immunized with immunogenic peptides were
chosen based on their higher reactivity towards A1-40 or A1-42. The Sp2/0Ag14 myeloma
cell line was fused with splenocytes from the selected immunized mice according to standard
protocols. Throughout the process, clone selection was done by sandwich ELISA based on the
capture of N-terminal biotinylated A1-40 or A1-42 (Anaspec, USA) on streptavidin-coated
plaques and their detection with hybridoma supernatant and goat anti-Fc antibodies (Sigma).
Hybridoma clones were selected based on their specific reactivity towards the relevant
biotinylated peptide and absence of reactivity against the other biotinylated peptide. Selected
antibody-producing hybridoma clones were sub-cloned twice and then frozen in liquid
nitrogen. Monoclonal antibodies were produced in vitro by collecting highly concentrated
supernatants. Specific anti-40 (6H7 clone) and anti-42 (12E8 clone) antibodies were selected,
amplified and purified on protein A Sepharose columns (GE Healthcare).
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Cell models
HEK293 cells that stably express wild type APP (APPwt) alone or together with the βsecretase BACE1 (APPwt+BACE1), or Swedish mutant APP (APPsw) were obtained and
cultured as described previously 1, 2.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Table S1
a
Injected
a
analyte
Immobilized
b
ligand
Ka (M-1s-1)
Kd (s-1)
KD (M)
Rmax (RU)
χ2
A1-40
4G8
1.89E+05
4.84E-03
2.56E-08
154.37
5.45
A11-40
4G8
1.94E+05
5.23E-03
2.69E-08
165.12
7.66
A17-40
4G8
2.82E+05
2.80E-02
9.95E-08
118.78
2.06
A1-42
4G8
5.93E+04
4.83E-03
8.15E-08
319.37
17.96
A11-42
4G8
1.19E+05
4.71E-03
3.96E-08
124.03
4.36
A17-42
4G8
A1-40
6H7
4.59E+05
1.15E-02
2.51E-08
99.43
6.83
A11-40
6H7
4.13E+05
1.03E-02
2.48E-08
109.2
8.71
A17-40
6H7
4.63E+05
1.16E-02
2.51E-08
87.69
6.21
A1-42
6H7
No association
A11-42
6H7
No association
A17-42
6H7
No association
A1-40
12E8
Very weak association
A11-40
12E8
Very weak association
A17-40
12E8
Very weak association
Very weak association
A1-42
12E8
1.94E+05
3.47E-04
1.79E-09
300.85
19.41
A11-42
12E8
3.16E+05
4.02E-04
1.27E-09
249
7.73
A17-42
12E8
5.79E+04
3.12E-04
5.39E-09
181.57
6.02
Analyte refers to the sample that was injected over the immobilized chip surface
b
Ligand refers to the sample that is covalently linked to the sensor chip surface
Table S1: Characterization of the 6H7 (anti-40) and 12E8 (anti-42) monoclonal antibodies
(mAbs) using the ProteOn array system and AβN-40/42 peptides.
Surface plasmon resonance (SPR) analyses were performed at 25°C using a ProteOn XPR36
instrument (Bio-Rad) with a GLM sensorchip (Bio-Rad). The 6H7 and 12E8 mAbs (HT-Mab
Sysdiag) were immobilized on the sensorchip via standard amine-coupling chemistry at a
density of ~10000 response units (RU). The surface was deactivated with 1M ethanolamine
hydrochloride solution. A control anti-Aβ (4G8, Covance, USA) mAb and an irrelevant (IRR)
mAb were immobilized at similar density on the chip to control non-specific binding and to
validate the experiment. Binding experiments were performed using PBS/0.005% Tween20 as
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
running buffer. The binding sensorgrams were recorded by injecting different concentrations
(from 3.125 to 200nM) of freshly prepared Aβ1-40 peptide (Anaspec, USA) in running buffer
over the immobilized antibody surface at a flow rate of 50µl/min for 4 min. The dissociation
profile was monitored for 10 min. The surface was regenerated with 0.85% phosphoric acid.
The activity of the immobilized antibody was not affected by the regeneration conditions and
the chip was used for further experiments. The binding kinetic constants were obtained using
a simple 1:1 Langmuir binding model. Data were analysed using the ProteOn Manager
software (Bio-Rad). After subtracting the background response obtained with the IRR
antibody, the association and dissociation phases were fitted simultaneously using the global
fit option. The calculated affinity constants (KD) were statistically validated by using the Chi2
value (< 10% Rmax).
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S1
Figure S1: The 6H7 and 12E8 mAbs allow the specific detection of AβN-40 and AβN-42
peptides, respectively. Supernatants of HEK293 cells that express wild type APP and
BACE1 were analysed by SELDI-TOF MS using PS20 chips pre-coated with the different
anti-Aβ monoclonal antibodies (black signal) or the irrelevant antibody (grey). The 6E10
(epitope 4-8 of A) and 4G8 (epitope 18-24 of A) antibodies captured all the A peptides.
The 6H7 and 12E8 antibodies did not recognize any other C-truncated peptide, even when
differing by only one amino acid
PS20 ProteinChip reactive arrays (Bio-Rad Laboratories, Hercules, US) were used for the
SELDI-TOF experiments. 2.5µl antibody solution (diluted to 0.4mg/mL in PBS) was added to
the chips that were then incubated at room temperature (RT) in a humidity chamber for 2h.
Excess antibodies were then removed and chips incubated in 20 µl of blocking buffer (10g/l
BSA in 100mM TRIS, pH 8) at RT in a humidity chamber for 30min. After removal of the
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
blocking buffer, a 20µl drop of sample (cell supernatant or CSF) was added to each spot and
chips were incubated at 4°C overnight. Excess sample was then removed and each spot
washed with 100 µl PBS/0.2% TRITON X100 three times, with 100µl PBS twice and with
100 µl MilliQ H2O once. Chips were air-dried and 1µl of matrix solution (20% α-Cyano-4hydroxycinnamic acid saturated in 50% (v/v) acetonitrile and 0.25% TFA) was applied to
each spot. Chips were air-dried, read with a ProteinChip Reader Series 4000 and analysed
with the ProteinChip System, Series 4000 software (Bio-Rad).
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S2
Figure S2: Assessment of the sensitivity of the Cter11 and Cter17 multiplexed assays using
synthetic peptides. Each panel represents the mean fluorescence intensity (FI) relative to the
AβN-40 or AβN-42 peptide concentrations, obtained in three independent experiments. The
sensitivities of the different assays are all below 10pg/ml.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S3
A
B
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S3: The two Cter assays allow the reliable measurement of N-truncated Aβ peptides in
complex fluids. The reproducibility of the 6H7/12E8/IRR triplex assays was evaluated using
HEK293 cell lines that over-express wild type or mutant APP and control CSF samples. AThe secretion of Aβ11-x peptides was strongly increased in the supernatant of HEK293 cells
that express wild type APP and BACE1 (APPwt+BACE1) and decreased in supernatants of
HEK293 cells that express Swedish mutant APP (APPsw) in comparison to cells that
express wild type APP (APPwt); the concentration of Aβ17-x peptides was decreased in the
supernatants of HEK293 APPwt+BACE1 or APPsw cells in comparison to APPwt cells.
Results are presented as the mean of three independent experiments using the same lot of
supernatants; B- Differential detection of Aβ11-x and Aβ17-x peptides in three control human
CSF samples (CSF A, B and C). Results are expressed as the concentration of the different
peptides in three independent experiments (Exp1, 2 and 3). The results of the CSF
measurements were reproducible (CV<20% for Aβ11-x measurement, CV<30% for Aβ17-x
measurement).
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S4
A
B
Figure S4: The 7H1 (anti-Aβ11x) and 8H5 (anti-Aβ17x) antibodies specifically detect Aβ1140 and Aβ17-40 peptides, respectively, in Cter multiplexed assays. The same
(6H7/12E8/IRR) triplex assay was used in all experiments for capturing the different AβN-40
peptides. A- The ability of the 6H7/7H1 and 6H7/4G8 sandwich assays to measure different
AβN-40 peptides (N=9-13) was compared; B- Comparison of the reactivity of the 6H7/8H5
and 6H7/4G8 sandwich assays towards AβN-40 peptides (N=15-19).
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S5:
A35000
35000
Ab11-40 + 0pg/ml Ab1-40
30000
Ab11-42 + 0pg/ml Ab1-42
30000
Ab11-40 + 100pg/ml Ab1-40
Ab11-40 + 1000pg/ml Ab1-40
25000
Ab11-42 + 250pg/ml Ab1-42
Ab11-42 + 500pg/ml Ab1-42
25000
Ab11-40 + 10000pg/ml Ab1-40
Ab11-42 + 1000pg/ml Ab1-42
20000
FI
FI
20000
15000
15000
10000
10000
5000
5000
0
0
1
10
100
1000
1
10000
10
100
1000
Ab11-42 (pg/ml)
Ab11-40 (pg/ml)
B35000
35000
Ab17-40 + 0pg/mL Ab1-40
30000
Ab17-42 + 0pg/mL Ab1-42
30000
Ab17-40 + 100pg/mL Ab1-40
Ab17-40 + 1000pg/mL Ab1-40
25000
Ab17-42 + 250pg/mL Ab1-42
Ab17-42 + 500pg/mL Ab1-42
25000
Ab17-40 + 10000pg/mL Ab1-40
Ab17-42 + 1000pg/mL Ab1-42
20000
FI
FI
20000
15000
15000
10000
10000
5000
5000
0
0
1
10
100
Ab17-40 (pg/ml)
1000
10000
1
10
100
1000
Ab17-42 (pg/ml)
Figure S5: Elevated amounts of full-length Aβ peptides do not interfere with the detection of
N-truncated peptides. The (6H7/12E8/IRR)/7H1 (for A11-x) (A) and (6H7/12E8/IRR)/8H5
(for A17-x) (B) triplex sandwich assays were used to measure the concentration of Ntruncated peptides in the presence of increasing concentrations of Aβ1-40/42 peptides; IRR:
irrelevant antibody.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S6
1.0
Sensitivity
0.8
0.6
0.4
0.2
T-Tau + P-Tau + A1-42 (AUC = 0.727)
T-Tau + A11-40 + A17-40 (AUC = 0.890)
0.0
1.0
0.8
0.6
0.4
1-Specificity
0.2
0.0
Figure S6: Testing the best CSF biomarker combinations for discriminating patients with MCI
from controls by using the mROC program. The current diagnostic test (AlzBio3 assay) is
represented by dotted lines (AUC=0.727) and the best mROC combination of biomarkers is
represented by a black line (AUC=0.890). The decision rules of these combinations were Z1 =
-0.003 Aβ42 + 0.016 T-Tau – 0.014 P-Tau and Z2 = 0.021 T-Tau – 0.027 Aβ11-40 + 0.023
Aβ17-40, respectively.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S7:
AA1-42
T-Tau
P-Tau
1000
100
-
600
400
*
*
200
-
Concentration (in pg/ml)
Concentration (in pg/ml)
800
150
100
50
-
80
60
-
40
20
200
0
CTRL
n=21
MCI≤1.5
n=9
0
MCI≥2
n=14
CTRL
n=21
MCI≤1.5
n=9
MCI≥2
n=14
CTRL
n=21
MCI≤1.5
n=9
MCI≥2
n=14
BA11-40
A11-42
250
-
50
*
Concentration (in pg/ml)
Concentration (in pg/ml)
300
*
200
150
100
50
**
-
40
30
20
10
0
CTRL
n=21
MCI≤1.5
n=9
CTRL
n=21
MCI≥2
n=14
MCI≤1.5
n=9
MCI≥2
n=14
CA17-40
A17-42
40
-
-
Concentration (in pg/ml)
120
Concentration (in pg/ml)
Concentration (in pg/ml)
**
-
100
80
60
40
20
0
-
30
*
-
20
10
0
CTRL
n=21
MCI≤1.5
n=9
MCI≥2
n=14
CTRL
n=21
MCI≤1.5
n=9
MCI≥2
n=14
Figure S7: Diagnostic value of the different CSF biomarkers for stratifying patients with MCI:
A- Aβ1-42, T-Tau or P-Tau levels were not significantly different between controls (CTRL)
and the MCI≤1.5 group (patients with a CDR-SB score≤1.5); B- A11-40 concentration was
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
significantly lower in both MCI subgroups (MCI≤1.5 or MCI≥2) than in controls; C- A17-x
peptide concentration was slightly higher in the MCI≤1.5 subgroup; -: p-value > 0.05; *: pvalue < 0.05; **: p-value < 0.01.
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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information
Figure S8:
17-40/11-40 ratio
(CDR-SB cut-off = 2)
17-40/11-40 ratio
(CDR-SB cut-off = 3)
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
p=0.05
C (n=21)
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
CDRSB≤3
(n=17)
CDRSB≥3.5
(n=6)
CDRSB≤1.5
(n=9)
CDRSB≤2
(n=12)
CDRSB≥2.5
(n=11)
17-40/11-40 ratio
(CDR-SB cut-off = 1)
p=10-3
C (n=21)
-2
C (n=21)
17-40/11-40 ratio
(CDR-SB cut-off = 1.5)
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
p=10
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
CDRSB≥2
(n=14)
p=10-4
C (n=21)
p=10-3
CDRSB≤1
(n=6)
CDRSB≥1.5
(n=17)
Figure S8: Diagnostic value of the A17-40/A11-40 ratio in MCI patients according to the
CDR-SB cut-off. The discrimination between the two groups of MCI patients is improved
when the CDR-SB cut-off is decreased. The A17-40/A11-40 ratio allows the molecular
characterization of these two subpopulations.
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Chevallier N, Vizzavona J, Marambaud P, Baur CP, Spillantini M, Fulcrand P et al.
Cathepsin D displays in vitro beta-secretase-like specificity. Brain Res 1997; 750(12): 11-19.
2.
Andrau D, Dumanchin-Njock C, Ayral E, Vizzavona J, Farzan M, Boisbrun M et al.
BACE1- and BACE2-expressing human cells: characterization of beta-amyloid
precursor protein-derived catabolites, design of a novel fluorimetric assay, and
identification of new in vitro inhibitors. J Biol Chem 2003; 278(28): 25859-25866.
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