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 (A31-40) or Fmoc-L-Ala (A33-42) 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 (A31-40) or Fmoc-L-Ile (A33-42), with HBTU (2-(1H-benzotriazol-1-yl)-

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 (Fmoc-

L-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, 15m, 300, 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-A31-40: 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 C-

KKKGS-A33-42 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

m, 300, 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-A33-42 and the C-KKK-PADRE-A31-40 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-A31-40 or BSA-A33-42 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 A1-40 or A1-42. 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 A1-40 or A1-42 (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 APP (APPwt) alone or together with the -

secretase BACE1 (APPwt+BACE1), or Swedish mutant APP (APPsw) were obtained and

cultured as described previously .

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Table S1

Table S1: Characterization of the 6H7 (anti-40) and 12E8 (anti-42) monoclonal antibodies

(mAbs) using the ProteOn array system and AN-40/42 peptides.

Surface plasmon resonance (SPR) analyses were performed at 25C 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-A (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 A1-40 peptide (Anaspec, USA) in running buffer

over the immobilized antibody surface at a flow rate of 50l/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 (K D) were statistically validated by using the Chi 2

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 AN-40 and AN-42

peptides, respectively. Supernatants of HEK293 cells that express wild type APP and

BACE1 were analysed by SELDI-TOF MS using PS20 chips pre-coated with the different

anti-A 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. 2.5l 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 l 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 20l drop of sample (cell supernatant or CSF) was added to each spot and

chips were incubated at 4C overnight. Excess sample was then removed and each spot

washed with 100 l PBS/0.2% TRITON X100 three times, with 100l PBS twice and with

100 l MilliQ H2O once. Chips were air-dried and 1l of matrix solution (20% -Cyano-4-

hydroxycinnamic 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

AN-40 or AN-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

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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 A 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 APP and control CSF samples. A-

The secretion of A11-x peptides was strongly increased in the supernatant of HEK293 cells

that express wild type APP and BACE1 (APPwt+BACE1) and decreased in supernatants of

HEK293 cells that express Swedish mutant APP (APPsw) in comparison to cells that express

wild type APP (APPwt); the concentration of A17-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 A11-x and A17-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 A11-x measurement, CV<30% for A17-x

measurement).

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Figure S4

Figure S4: The 7H1 (anti-A11x) and 8H5 (anti-A17x) antibodies specifically detect A11-

40 and A17-40 peptides, respectively, in Cter multiplexed assays. The same (6H7/12E8/IRR)

triplex assay was used in all experiments for capturing the different AN-40 peptides. A- The

ability of the 6H7/7H1 and 6H7/4G8 sandwich assays to measure different AN-40 peptides

(N=9-13) was compared; B- Comparison of the reactivity of the 6H7/8H5 and 6H7/4G8

sandwich assays towards AN-40 peptides (N=15-19).

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Figure S5:

A-

B-

Figure S5: Elevated amounts of full-length A peptides do not interfere with the detection of

N-truncated peptides. The (6H7/12E8/IRR)/7H1 (for A11-x) (A) and (6H7/12E8/IRR)/8H5

(for A17-x) (B) triplex sandwich assays were used to measure the concentration of N-

truncated peptides in the presence of increasing concentrations of A1-40/42 peptides; IRR:

irrelevant antibody.

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Figure S6

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 A42 + 0.016 T-Tau 0.014 P-Tau and Z2 = 0.021 T-Tau 0.027 A11-40 + 0.023

A17-40, respectively.

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Figure S7:

A-

B-

C-

Figure S7: Diagnostic value of the different CSF biomarkers for stratifying patients with MCI:

A- A1-42, T-Tau or P-Tau levels were not significantly different between controls (CTRL)

and the MCI1.5 group (patients with a CDR-SB score1.5); B- A11-40 concentration was

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

significantly lower in both MCI subgroups (MCI1.5 or MCI2) than in controls; C- A17-x

peptide concentration was slightly higher in the MCI1.5 subgroup; -: p-value > 0.05; *: p-

value < 0.05; **: p-value < 0.01.

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Figure S8:

Figure S8: Diagnostic value of the A17-40/A11-40 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 A17-40/A11-40 ratio allows the molecular

characterization of these two subpopulations.

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Abraham et al., N-truncated A peptides for MCI diagnosis; supplementary information

Supplementary bibliography

1. 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(1-2):
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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