Analysis of Amyloid-β Peptides in Cerebrospinal ... - ACS Publications

Feb 11, 2011 - LVR-Hospital, Essen Department of Psychiatry and Psychotherapy, University of Duisburg-Essen, Virchowstrasse 174, D-45147 Essen,...
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Analysis of Amyloid-β Peptides in Cerebrospinal Fluid Samples by Capillary Electrophoresis Coupled with LIF Detection Romain Verpillot,† Hermann Esselmann,‡ Mohamad Reza Mohamadi,§ Hans Klafki,‡ Florence Poirier,|| Stefan Lehnert,^ Markus Otto,^ Jens Wiltfang,‡ Jean-Louis Viovy,§ and Myriam Taverna*,† †

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Univ. Paris-Sud, UMR-CNRS 8612, Laboratory of Proteins and Nanotechnologies in Separation Sciences, 92296, Faculte de Pharmacie, Ch^atenay-Malabry, France. ‡ LVR-Hospital, Essen Department of Psychiatry and Psychotherapy, University of Duisburg-Essen, Virchowstrasse 174, D-45147 Essen, Germany § UMR 168, Institute Curie/CNRS/Universite Pierre et Marie Curie, Paris, France Proteomic Platform of IPSIT, Faculte de Pharmacie, Ch^atenay-Malabry, France ^ University of Ulm, Department of Neurology, Steinh€ovelstr. 1, 89075 Ulm, Germany ABSTRACT: We report a CE-LIF method for the separation and detection of five synthetic amyloid-β peptides corresponding to an important family of CSF-biomarkers in the context of Alzheimer disease (AD). The presumed most relevant peptides (Aβ1-42, Aβ1-40, and Aβ1-38) that may support the differentiation between AD and healthy patients or other dementias were successfully detected in CSF by incorporating an immunoconcentration step prior to CE analysis of derivatized peptides. We labeled the Aβ peptides with a fluoroprobe dye before CE-LIF analysis. This reagent reacts with the amino groups of lysine residues and produced mostly ditagged Aβ peptides under the proposed experimental conditions. The labeling reaction displayed similar efficiency with each one of the five different synthetic Aβ peptides that were tested. The limit of detection of the CE-LIF method approached 280 attomoles of injected synthetic labeled Aβ peptides. We obtained excellent correlation between peak areas and peptide concentrations from 35 nM to 750 nM. For the detection of Aβ peptides in human CSF samples, we enriched the peptides by immunoprecipitation prior to the CE-LIF analysis. The comparison of the CE-LIF profiles obtained from CSF samples from 3 AD patients and 4 non-demented control subjects indicated noticeable differences, suggesting that this method, which relies on a multibiomarker approach, may have potential as a clinical diagnostic test for AD.

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myloid-β 1-42 (Aβ1-42) peptide is a major component of senile plaques deposited in the brain of individuals with Alzheimer’s disease (AD).1 Aβ1-40 peptide and its C-terminal elongated form Aβ 1-42 are produced from the amyloid precursor protein (APP) through sequential proteolytic cleavages by β and γ-secretases.2 Other shortened Aβ peptides such as Aβ137, Aβ1-38, and Aβ1-39 were discovered in the brain and cerebrospinal fluid (CSF), indicating that Aβ constitutes a large family of peptides with length variations.3-5 A selective reduction of the Aβ peptide 1-42 level in CSF is considered as a robust biomarker of AD.6-9 However, several other neurodegenerative or dementia diseases are also associated with low level of Aβ1 -42 in CSF, notably, dementia with Lewy body (DLB), Parkinson's disease (PDD), or Creutzfeld-Jacob disease (CJD).10 Several authors11-14 proposed the measurement of the Aβ1-42/ Aβ1-40 ratio to improve the diagnosis of AD. These measurements are typically performed with single ELISA tests. However, a high variability of the results are frequently observed and in several cases, it is not possible to discriminate AD from other dementias using current assays. Others attempts to quantify these two peptides in biological fluids have included a combination of immunoprecipitation and MALDI-TOF analysis.15,16 The analysis of CSF for multiple biomarkers and, in particular, the combined and simultaneous quantitation of the amyloid quintet (Aβ1-42, Aβ1-40, Aβ1-39, Aβ1-38, and Aβ1-37), represents, in r 2011 American Chemical Society

this context, a promising approach for an improved early and differential dementia diagnosis. Using a urea-based SDS-PAGE with Western blot developed by Wiltfang et al.14 and Bibl et al.17 observed that Aβ peptide patterns in CSF, and especially the ratio between Aβ1-42 and 1-38,18-20 varies in a disease-specific manner between CJD, DLB, and PDD. We recently reported a fast CZE-UV method for separation of the five amyloid peptides.20 The sensitivity of this method, however, was not sufficient for measurements in body fluids. In the current work, we have developed a method for fluorescently labeling Aβ peptides prior to CE separation and LIF detection. The aim of our work was therefore to provide a new tool addressing especially the sensitivity limitation but also the separation performance required for a multibiomarker approach, as a step toward an early and differential diagnosis of AD. In an attempt to develop a diagnostic method, microfluidic chips were used to separate the fluorescently labeled Aβ peptides.21 Although this method was fast, the peptides could not be baseline resolved due to the short channel length. This generated potential inaccuracies for the analyses used to develop the Received: October 27, 2010 Accepted: January 14, 2011 Published: February 11, 2011 1696

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Analytical Chemistry concentration ratios. We decided that better resolution could be obtained in CE albeit in a longer time frame which was still much shorter than the duration of a full SDS-PAGE-Western blot protocol. We report here labeling and separation protocols and conditions able to (i) maintain the solubility of the peptides and their proper conformation for derivatization and analysis, avoiding their adsorption to the dynamically modified capillary surface; (ii) ensure an efficient and fast (5 min) labeling of very diluted peptides in the presence of a strong excess of other polypeptides and proteins, and (iii) produce a single dominant tagged species per peptide. The method was validated in terms of response linearity, limit of detection and repeatability. Finally, the procedure was applied to a small number of real CSF samples from three patients suffering from AD and four nondemented patient controls.

’ MATERIAL AND METHODS Chemicals, Reagents and Samples. Amyloid peptides were purchased from Anaspec (Le Perrey en Yvelines, France), and R-peptide (Bogart, GA, United States). Sodium hydroxide (1 M) was obtained from VWR (Fontenaysous Bois, France). Boric acid (H3BO3) ammonium hydroxide 28.1% (m/V), Thiourea and Diaminobutane chloride (DAB) were obtained from Sigma (St. Louis, MO, United States). DMSO 99.9% purity for analysis was obtained from Sigma (St. Louis, MO, United States). The Fluoprobe 488 NHS, Alexa fluor 488 NHS, FAM X-SE NHS, and 5-FITC were obtained from Interchim (Montluc- on, France). All buffers were prepared with deionized water and were filtered through a 0.22 μm membrane before use. Apparatus and Material. Uncoated capillaries were from Phymep (Paris, France). The studies have been performed using a Beckman Coulter PA 800 ProteomLab equipped with either a Photodiode array detection (DAD) or coupled with LIF detection. Data acquisition and instrument control were carried out using Karat 7.0 software. Deionized water used in all experiments was purified using a Direct-Q 3 UV purification system (Millipore, Milford, MA, USA). pHs of buffer solutions were adjusted with inoLab WTW series pH 730 pH meter. Buffer ionic strength (IS) calculations were performed using the Phoebus program (Analis, Orleans, France). Methods. Dissolution and Storage of the Peptide Samples. Dissolution medium and storage conditions are critical parameters for amyloid peptides, which are prone to aggregation or fibrillogenesis. Standard Aβ1-42 was dissolved in 0.16% (m/V) ammonium hydroxide aqueous solution. All other amyloid peptides were dissolved in 0.10% (m/V) ammonium hydroxide. Aliquot solutions of individual peptides were prepared at 2 mg/mL and stored at -20 C. Labeling Reaction. The five peptides from aliquot solutions were mixed and diluted 2-fold in borate buffer (pH 10.5, IS 40 mM), then, lyophilized to remove the ammonium hydroxide. The powder was subsequently dissolved in the same borate buffer to reach the desired concentration of the peptides and a final ionic strength over 40 mM. Finally, 4 μL of Fluoprobe from stock solution at 10 mg/mL in DMSO was added and incubated 5 min at RT. This generic reaction was also used for FAM X-SE, Alexa Fluor, and 5-FITC labeling reactions. However, the 5-FITC was incubated with peptides for 12 h at 8 C instead of 5 min at RT. The reaction products were analyzed directly by CZE without any treatments or clean up.

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Monitoring of the labeling reaction was performed by CZE but in the UV detection mode (DAD detector). For these experiments, the concentration of peptides in the mixture was kept constant and different excesses of Fluoprobe (1, 4, and 20) were employed. To dissolve Aβ1-42 and to maintain a monomeric conformation, the addition of NH4OH in the lyophilized samples is recommended by the suppliers. We observed that the presence of trace amounts of ammonium hydroxide decreased the yield of the labeling reaction in a random fashion (data not shown). Therefore, all of the standard peptides had to be first lyophilized under alkaline medium to efficiently remove ammonia and then dissolved in water or in the buffers. Capillary Electrophoresis Conditions. New capillaries were pretreated by applying pressures of 138 kPa to the capillary inlet and using the following sequence: 0.1 M NaOH for 5 min, 1 M NaOH for 5 min, and then water for 5 min. After this pretreatment, five successive cycles were carried out to stabilize the silica surface: One cycle entailed one rinsing step with the running buffer (10 min) then voltage application at 30 kV (for 15 min), followed by one rinse with water (10 min) and 0.1 M NaOH (5 min). The in-between rinsing run was carried out by pumping deionized water through the capillary for 5 min, DMSO/water (50:50) and then water 3 min each using reversed flow, and finally the running buffer for 10 min. The samples were introduced into the capillary by hydrodynamic injection for 5 s and at 3.4 kPa (corresponding to ∼8 nL of injected sample). The capillary was thermostatted at 25 C and the samples were maintained at 10 C by the storage sample module of the PA 800. The peptides were detected using LIF detection (a 3.5 mW argon-ion laser having an excitation at 488 nm; emission was collected through a 520 nm band-pass filter). The separations were carried out at 30 kV with positive polarity at the inlet and using borate buffer pH 9 at IS 40 mM, containing 3.25 mM of DAB to ensure a dynamic coating. The running electrolyte was renewed after every run and prepared daily. Calibration Curve. Standard solutions containing the five peptides at equal concentrations were employed to evaluate the linearity of the method. The concentrations tested ranged from 35 to 750 nM. The mixtures were labeled using the protocol described in the “Labeling Reaction” section. The analyses were carried out in triplicate for each concentration level and the calibration curves were calculated by means of the least-squares method. CSF Sample Collection. All CSF samples analyzed in this work were taken from patients attending the department of Neurology in Ulm (University of Ulm, Department of Neurology) in 2008 and 2009. Collection and analysis of CSF samples was approved by the Ethics Committee in Ulm. All individuals gave written informed consent to their participation in the study and underwent a clinical, neurological, and neuroradiological examination, as well as a short neuropsychological screening, including Mini-Mental State Examination to investigate global cognitive functioning. If deterioration were suggested, then a detailed psychometric test battery covering executive functions, memory, constructional abilities, premorbid verbal intelligence, and depression was administered to assess more specifically for cognitive impairment.22,23 CSF was taken, aliquoted within 2 h and stored at -80 C. Immunoprecipitation and Labeling of CSF. Magnetic microparticles (Dynabeads M-280 Covance, Emeryville, CA) were coated with monoclonal antibody anti-Aβ 6E10 (Covance, Emeryville, 1697

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Analytical Chemistry CA). A total of 100 μg of anti-Aβ antibodies were coupled to 1 mg of magnetic microparticles, following the procedure suggested by the supplier. A total of 250 μL of CSF was added to 250 μL of borate buffer pH 10.5, IS 80 mM then 10 μL of Fluoprobe was added to this diluted CSF; at this stage, the pH value of the sample mixture was checked to be 10.5. This solution was mixed and incubated for 5 min in dark. A 500 μL portion of RIPA buffer (which was composed of 50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM protease inhibitor; pH adjusted to 7.4 with NaOH) and 25 μL of magnetic microparticles coated with the monoclonal antibody 6E10 were added to the sample mixture. Samples were incubated under rotation for 15 h at 4 C. After mixing, the sample was washed first with RIPA buffer, then with 50 mM Tris/HCl, pH 7.4; and last with deionized water. For the elution step, we used the same aqueous solution of ammonium hydroxide as for standard peptides: 400 μL of 0.16% NH4OH. The eluted Aβ peptides were fully lyophilized in polypropylene tubes and reconcentrated in 2 μL of 0.16% NH4OH prior to CZE-LIF analysis. SDS-PAGE of Aβ Peptides and Western blotting. For the separation and visualization of Aβ peptides, the urea version of the bicine/sulfate SDS-PAGE and the semidry Western blotting method according to Wiltfang et al. were used.14 The composition of the separation gel applied for the analysis of Aβ peptides was 10%T%/5%C/8 M urea and the gel thickness was 0.5 mm. The gels were run at room temperature for 1 h at a constant current of 25 mA/gel using the MiniProtean II electrophoresis unit (BioRad). After blotting onto PVDF membranes, immunostaining was performed with the monoclonal antibody 1E8 (Nanotools) overnight (14 h) at 4 C. Then, the membranes were washed and incubated with a biotinylated antimouse antibody (Vector Laboratories) for 1 h. Following a further washing step, horseradish peroxidase-coupled streptavidin was added for 1 h. The membranes were washed and finally, developed with ECL-Plus solution (Amersham Pharmacia) according to the manufacturer’s instructions, using a CCD camera (INTAS, Goettingen, Germany). To perform the SDS-PAGE of labeled CSF, the CSF samples were labeled the same way as synthetic peptides: the CSF samples (50 or 100 μL) were diluted 2-fold in borate buffer (pH 10.5, IS 80 mM) to reach 100 or 200 μL as a final volume after addition of 2 or 4 μL of Fluoprobe (from stock solution at 10 mg/mL in DMSO). MALDI-TOF MS Analysis. MALDI-TOF analyses of the derivatized samples were carried out using a Voyager DE-STR MALDI-TOF mass spectrometer (Perseptive Applied Biosystems) equipped with a 337 nm nitrogen laser. Samples were mixed with saturated solution of sinapinic acid (SA) in 50% ACN/0.1% Formic Acid and deposed on a MALDI sample plate. All MS spectra were acquired in positive ion linear mode using four proteins of known masses (Sigma-Aldrich, Steinheim, Germany) as close external calibration standards. The peptide mass profiles obtained by MALDI-TOF MS were analyzed using the Data Explorer software (Applied Biosystems).

’ RESULTS AND DISCUSSION Optimization of the Prederivatization Reaction. To improve the detection sensitivity of our original UV-CE method, we chose to fluorescently label primary amines of the amyloid peptides. The five amyloid peptides investigated here possess

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Figure 1. CZE-LIF profile of the five amyloid peptides derivatized with Fluoprobe (A) or Alexa fluor (B), FAM-X-SE (C), and 5-FITC (D) as the tagging agents. F is a peak from the fluorophore. The peptide concentration was 100 nM except for the Fluoprobe derivatization (50 nM). Brackets indicate regions corresponding to the migration of tagged Aβ peptides. CE conditions; BGE: borate buffer pH 9; IS 40 mM with 3.25 mM of DAB, Detection: LIF (λ exc 488 nm).

three primary amines each, one R-amino group (NH2-terminus) and 2 ε-amino groups at the lysine side chains. In theory, seven different tagged species can be produced from each one of the peptides. Two different approaches were explored aiming for limited complexity of the peptide mixtures obtained after the labeling reaction, namely the (i) specific labeling of the R-NH2 group (pKa ≈ 8.5) by selecting a near- neutral pH for the reaction medium and (ii) full labeling using alkaline pH favoring the reaction on all available amines including the lysine side chain ε-amino groups (pKa ≈ 10.54). The first approach failed while the full labeling approach in a borate buffer at pH 10.5 was successful. According to Giacomelli et al. (2005),24 at pH 10.5, the amyloid peptides adopt a random coil helix, whereas below this pH value, they spontaneously twist into β-sheet polymer structures. In addition to increasing the nucleophilic properties of the primary amino groups, the alkaline reaction pH was also expected to impact the conformation of the peptide and thereby improve accessibility of amines to the reagent. High pH values have the advantage to stabilize a favorable conformation of the peptide for derivatization and to avoid the aggregation of Aβ1-42.24 Four different fluorophores were investigated: FAMX-SE (Mw, 586.6), Alexa fluor (Mw, 643), Fluoprobe (Mw, 981) and FITC (Mw 389.4) were used to label the mixture of five synthetic peptides (Aβ1-42, Aβ1-40, Aβ1-39, Aβ1-38, and Aβ1-37) at pH 10.5 and under the same concentration conditions. Except for FITC, these fluorophores bear the same reactive function (N-hydroxyl-succinimidyl ester) but carry different fluorescent scaffolds. The reaction products were directly analyzed by CZELIF using a dynamic coating with DAB (diaminobutane chloride) (Figure 1). The role of DAB in preventing peptide adsorption to the capillary wall and ensuring a higher separation resolution was demonstrated in a previous work.20 Using Alexa fluor, Fluoprobe, and FITC, the labeled peptides migrated as two groups of peaks. FAM, bearing a long linker chain, led to more than 11 peaks indicating a higher number of heterogeneously tagged species. With 5-FITC, it was possible to obtain, in the first 1698

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Figure 2. (A) CE-UV monitoring of the derivatization reaction of mixture of the five peptides (conc: 20 μM) with two different concentrations of tagging reagent (1 and 4) and (B) MALDI-TOF analysis of the reaction products obtained after Aβ1-38 derivatization with Fluoprobe under different concentrations of the tagging agent. The peak at m/z: 4132 corresponds to the unlabeled Aβ1-38; the one at m/z: 4705 to the monotagged, Aβ1-38*; and m/z: 5728 to the ditagged Aβ1-38**. CE conditions: BGE: borate buffer pH 9; IS 40 mM with 3.25 mM of DAB, Detection: UV at 190 nm.

group of peaks, the five peptides with one species per peptide. In contrast, Alexa fluor produced numerous and not fully resolved peaks. Fluoprobe led to a very satisfactory profile, with the second group of peaks containing only five peaks, suggesting one labeled species per peptide. The observed differences between the four reagents, regarding the number of labeled species, might be related to the steric hindrance induced by the structure of the fluorophore and the proximity of the lysine residues in all peptides. Characterization of the Labeling Reaction Yield. In order to be able to ultimately compare the relative proportions of the five amyloid peptides between different CSF samples, the tagging efficiency has to be equivalent for the five different peptides. To optimize the reaction with Fluoprobe and to confirm comparable labeling efficiencies, we monitored the reaction by CZE in UV detection mode (Figure 2A). Under these conditions, both unlabeled and labeled peptides were detected. Different concentration ratios between Fluoprobe and peptides were compared. With a 4-fold (molar) excess of Fluoprobe, the unlabeled peptides completely disappeared, as well as the first group of peaks (Figure 2A). Only the second group of peaks was observed indicating that the reaction was complete. To elucidate the number of Fluoprobe molecules per molecule of peptide, MALDI-TOF analyses were performed on the tagged Aβ1-38 at different ratio of Fluoprobe to Aβ1-38 concentration. The mass spectra (Figure 2B) show three distinct species: one at m/z 4132, which corresponds to the mass of the

unlabeled peptide; the others at 4705 and 5278 correspond to the monotagged and ditagged species, respectively. In addition, the mass increment of 573 g/mol (and 1146, respectively), confirm this assignment. Even with the presence of large excess of Fluoprobe (20), no peak was detected at the m/z expected for the tritagged peptide. We therefore concluded that the first group of peaks appearing in Figure 2A (Tm around 14 min) corresponded to the monotagged species while the second group (Tm around 24 min) referred to ditagged species. When the labeling was done with a 1 or 4 molar ratio of fluophore to peptide, the relative peak areas observed with CE-LIF for the five ditagged peptides (Aβ1-37**, Aβ1-38**, Aβ1-39**, Aβ1-40**, Aβ1-42**) were 14, 18, 27, 19, and 22%, respectively, and were found to be similar to that estimated for the corresponding mixture of the unlabeled peptides. From these results, we concluded that all of the peptides within the mixture were quantitatively labeled with an excess of labeling reagent with similar efficiencies, and that optimized conditions (i.e., 4 excess of fluorophore) led to a quasi-complete reaction producing mainly ditagged species. Attempts to produce tritagged species by varying derivatization conditions (e.g., with a large excess of fluorophore) were unsuccessful. This suggested that the proximity between Lys 16 and Lys 28 may have precluded the covalent attachment of two fluorescent tags. The first attachment of one Fluoprobe molecule on one of the two side-chain amino-group may lead to steric 1699

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Figure 3. Proposed model of the derivatization reaction of the five amyloid peptides with the Fluoprobe.

Table 1. Linearity of the Response and Reproducibility of Migration Times and Peak Areas of the Whole Method peptides

calibration curve (r2)

RSD migration times (%)

RSD peak areas (%)

Aβ1-42

0.996

0.69

1.76

Aβ1-40

0.997

0.72

3.46

Aβ1-39

0.999

0.73

3.54

Aβ1-38

0.997

0.76

4.3

Aβ1-37

0.997

0.77

4.9

constrains regarding the fixation of another molecule on the second intrachain lysine. In addition, the peaks corresponding to the ditagged species appear relatively broad (4 broader than the unlabeled peptide ones). Even if we take into account the increase of axial diffusion for later migrating species, the broadness of peaks of ditagged peptides can suggest the comigration of 2 isoforms per peptide under one peak, this would be in agreement with the most probable model of derivatization reaction of the five amyloid peptides with the Fluoprobe proposed in Figure 3. Validation of the CE-LIF Method. The CE-LIF method was finally validated in terms of linearity and repeatability, and the limits of detection for the five Aβ peptides were estimated. Five mixtures containing the five synthetic peptides were labeled independently on the same day and analyzed by CE-LIF. The RSDs of the migration times were less than 0.77% and those of the peak areas less than 4.9% (Table 1). Very satisfactory resolutions between adjacent synthetic Aβ peptides were found, with values ranging from 1.4 to 2.5. A resolution of 2.5 was attained between Aβ1-39 and Aβ1-38 by this CE-LIF method while these peptides could not be distinguished by the CE-chip method previously developed by our group.21 A key factor to reach good repeatability was the use of an aqueous solution of DMSO to rinse the capillary and to efficiently remove adsorbed Fluoprobe from the inlet of the capillary.

The linearity of the detection was assessed by triplicate analyses of solutions containing the mixture of the five peptides in concentrations ranging from 35 to 750 nM. The determination coefficients ranged from 0.998 to 0.995 showing an excellent linearity of response compatible with a fully quantitative method. The LOD (estimated at a S/N ratio equal to 3) with regard to the synthetic Aβ peptides was estimated to be close to 35 nM and therefore to the concentration range of the Aβ peptides found in the CSF (around 1 nM of total Aβ peptides). Throughout, it should be noted that, in commercial samples, a substantial fraction of the synthetic Aβ peptides may not be present as monomer upon receipt. Thus, the determination of the absolute concentration of one given monomeric peptides in biological samples may not be accurate, especially if the quantification is based on the use of these external peptide standards. Nevertheless, the method, which displays excellent response linearity, allows for accurate relative concentration determination and for comparison of samples with regard to their relative proportions of different Aβ variants. Application to CSF Samples. The optimized conditions for the derivatization of synthetic Aβ peptides were applied to the direct labeling of CSF samples. In order to check the efficiency of the labeling reaction with regard to the Aβ peptides in the CSF samples, we employed SDS-PAGE/Western blot analysis as a control method. The fluorescence labeled synthetic Aβ peptides displayed noticeable shifts in their electrophoretic mobilities on urea-SDS-PAGE as compared to the unlabeled peptides (Figure 4A). When a CSF sample was subjected to the same reaction, the intensity of the peptide bands corresponding to unlabeled Aβ was substantially decreased (Figure 4B, lane 5) as compared to the corresponding bands obtained with underivatized CSF (lanes 3 and 4). Moreover, new Aβ peptide bands were observed in low intensities, presumably representing the labeled peptides as judged from the position in the gel of Figure 4, lane 5. The low intensity 1700

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Figure 4. Monitoring by SDS-PAGE/Western blot of derivatized amyloid peptides from real CSF samples before (B) and after (C) immunocapture in comparison to the analysis of the labeled synthetic peptides (A). Lane 1: mixture of unlabeled standard peptides at 7.5 nM each; Lane 2: the mixture of synthetic labeled peptides at 55 nM after derivatization with Fluoprobe; Lanes 3 and 4: CSF samples of patients suffering from AD; Lane 5: CSF sample of patients suffering AD after derivatization; Lane 6: mixture of standard peptide Aβ1-42 (0.170 nM), Aβ1-40 (0.350 nM) and Aβ1-38 (0.350 nM); Lane 7: Elution Fraction of immunocaptured and labeled CSF from nD patient; Lane 8: Blank of elution fraction (same conditions as lane 7 but without CSF); Lane 9: mixture of unlabeled synthetic peptides: Aβ1-42 (0.170 nM), Aβ1-40 (0.350 nM), and Aβ1-38 (0.180 nM); and Lane 10: mixture of unlabeled synthetic peptides: Aβ1-42 (0.330 nM), Aβ1-40 (0.690 nM), and Aβ1-38 (0.360 nM).

Figure 5. Detection of Aβ peptides 1-42, 1-40, and 1-38 by CE-LIF in CSF from nondemented subjects (nD) (a) and patient suffering AD (b). Blank of immunocapture (c) The peak appearing at 19 min in profile (c) corresponds to a spike. CE conditions as in Figure 2.

may be attributed to the sample preparation for SDS-PAGE analyses, which requires temperatures as high as 95 C that can lead to partial destruction of the fluoroprobe. The degradation of the fluorophore attached to the peptides can generate very reactive species that can bind covalently to other molecules or proteins available rendering the detection of labeled peptides in the range of the molecular masses investigated not possible. To improve the recovery of labeled Aβ peptides from CSF, we employed immunoprecipitation prior to the SDS-PAGE/

Western blot analysis. After the immunocapture on magnetic beads carrying immobilized mAb anti-Aβ 6E10, we found bands at the same position as that with the derivatized CSF prior to the immunocapture (Figure 4C, lane 7). Figure 5 displays the CE-LIF profile obtained from 2 CSF samples subjected to fluorescence labeling followed by immunocapture. One of the two CSF samples was collected from an AD patient and the other from a nondemented subject (nD). The profiles suggested the presence of peaks corresponding to 1701

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Table 2. Different Ratios Representing Relative Peptide Concentrations, Estimated by CE-LIF from Three CSF from Patients Suffering AD and Four Other CSF from Non-Demented Subjects ratio of peptide (corrected areas) AD/nD

1-42/1-40

1-42/1-38

(1-42/1-42 þ 1-40 þ 1-38)  100

nD

0.32

0.94

19.3

nD

0.20

1.10

14.3

nD

0.41

0.35

15.9

nD

0.27

0.33

13.1

AD

0.16

0.26

8.9

AD AD

0.25 0.08

0.12 0.09

7.3 4.0

the labeled Aβ peptides Aβ1-42, Aβ1-40, and Aβ 1-38 which were not present in the blank. The very sharp peak (spike) appearing in the blank was supposed to be microbeads, used to performed the immunocapture of peptides that were not fully removed after the magnetic sequestration. The identification of the peptides was confirmed by spiking the CSF samples with synthetic Aβ peptides. The relative proportion of the three detected Aβ-peptides appeared to be different between the two samples. In addition, in the nondemented reference, we noticed more intense additional peaks, in the region between 17.5 and 19 min. Further studies will be required to identify these compounds. To analyze whether the observed differences can be also observed in additional CSF samples, two more AD and three more nD samples were tested. When comparing the CE-LIF profiles obtained for CSF from three AD patients with those from four nondemented subjects, a clear qualitative distinction could be observed. The calculated ratios between Aβ1-42 and Aβ 1-38 were higher in the four nD CSF samples than in the three AD samples (Table 2). In the small number of CSF samples analyzed herein, the Aβ1-42/Aβ1-38 ratio was found to allow for a better discrimination between AD patients and nD controls than Aβ1-42/Aβ1-40 ratio. Interestingly, all of the nD profiles presented higher Aβ1-42/(Aβ1-40 þ Aβ1-42 þ Aβ1-38) ratios than the three AD samples. Indeed, as displayed in Table 2, the ratios between the Aβ1-42 to the sum of the three peptides ranged from 13.1% to 19.3% for nD samples while it was between 4.0 and 8.9% for AD ones. To estimate the relevance of these differences, we have evaluated the repeatability of the whole method by performing three distinct immunocaptures, derivatizations, and CE analyses of the same CSF sample from one nD patient. The enriched and labeled CSF fractions produced RSD, in terms of the ratio of the Aβ1-42 to the sum of the three peptides, of 11.8%. This value highlights the pertinence of the preliminary discrimination between AD and nD patients with this small number of CSF samples. Although our findings need to be confirmed with a larger sample size, we speculate that our CELIF approach may prove to be of potential interest for supporting the clinical AD diagnosis.

’ CONCLUSIONS For the first time, we have achieved separation by CE-LIF of five labeled synthetic amyloid peptides, which we consider relevant biomarkers for the diagnosis of AD. The labeling method here was shown to produce high and constant derivatization efficiency for synthetic Aβ peptides. In combination with

immunoprecipitation, the method was successfully applied to the detection of Aβ peptides in CSF samples keeping a high resolution between the peptides Aβ1-42, Aβ1-40, and Aβ1-38. Our CE method therefore allowed detection and quantification of the three most concentrated peptides from the CSF under physiological and pathological conditions, which was not possible with the MCE method we previously reported. We compared the relative proportions of the Aβ peptides forms (Aβ1-42, Aβ1-40, Aβ1-38) in CSF samples from three AD patients and four nondemented subjects and observed preliminary evidence for noticeable group differences. Thus we conclude that this method may turn out to have the potential to improve the diagnostic specificity of AD by multi marker approach: The separation of clinically relevant peaks in the electrophoregrams and the linearity of their amplitudes avoid cross correlation and allow for a more quantitative evaluation of concentration ratios, as compared to monoplex immunoassays. As compared to SDSPAGE/Western Blot, CE-LIF considerably reduces manpower and experimental time, and is also more easily amenable to automation and quantitative analysis, thus facilitating the clinical development of a novel tool to support the differential diagnosis of neuropsychiatric disorders.

’ AUTHOR INFORMATION Corresponding Author

*Fax: 00 (33) 146835944. E-mail: [email protected].

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’ NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on February 11, 2011. An Acknowledgment was added, and corrections were made to the names of the second and ninth authors. The corrected version was reposted on February 28, 2011.

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