Microchip Electrophoresis Profiling of Aβ Peptides in the

Aug 19, 2010 - UMR 168, Curie Institute/CNRS/Université Pierre et Marie Curie, Paris, France, Department of Biological and. Biochemical Sciences, Uni...
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Anal. Chem. 2010, 82, 7611–7617

Microchip Electrophoresis Profiling of Aβ Peptides in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease Mohamad Reza Mohamadi,† Zuzana Svobodova,‡ Romain Verpillot,§ Hermann Esselmann,| Jens Wiltfang,| Markus Otto,⊥ Myriam Taverna,§ Zuzana Bilkova,‡ and Jean-Louis Viovy*,† UMR 168, Curie Institute/CNRS/Universite´ Pierre et Marie Curie, Paris, France, Department of Biological and Biochemical Sciences, University of Pardubice, 53210 Pardubice, Czech Republic, Faculte´ de Pharmacie, UMR 8612-LPNSS, University of Paris sud 11, Chatenay Malabry, France, Department of Psychiatry and Psychotherapy, LVR-Hospital, University of Duisburg-Essen, Virchowstrasse 174, D-45147 Essen, Germany, and Department of Neurology, University of Ulm, Steinho¨velstrasse 1, 89075 Ulm, Germany The preferential aggregation of Aβ1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer’s disease. This is accompanied by a relative reduction of the concentration of Aβ1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer’s disease. Here, we describe a microchip gel electrophoresis method in polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aβ peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aβ peptides (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ142) was achieved, and relative abundance of Aβ1-42 to Aβ1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aβ peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer’s disease. Aβ peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aβ were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aβ peptides (Aβ1-40 and Aβ1-42) in the CSF samples. Neurodegenerative diseases (ND) are a major issue in human health. Alzheimer disease (AD), in particular, is one of the most common age-related neurodegenerative disorders and is becoming a paramount societal issue with the aging of the population. The reliable diagnosis of AD in the early stages has become all the more important, for several reasons. First, the disease has a very long asymptomatic evolution, probably over 10 years, during which neurological damages are, nevertheless, irreversible. Second, neuroprotective treatments able to retard the disease’s progression * Corresponding author. Phone/Fax: +33 1 40 51 06 36. E-mail: [email protected]. † Curie Institute/CNRS/Universite´ Pierre et Marie Curie. ‡ University of Pardubice. § University of Paris sud 11. | University of Duisburg-Essen. ⊥ University of Ulm. 10.1021/ac101337n  2010 American Chemical Society Published on Web 08/19/2010

will hopefully be available in the future, but there is no shortterm hope of treatments able to reverse the existing damages. Finally, the development of specific treatments raises the need for the differential diagnosis between different NDs. At the present time, diagnosis of AD is mainly based on clinical and neurological symptoms. At early stages, however, different forms of dementia are not easy to distinguish, and the differentiation between dementia and depression might also be difficult to obtain. Thus, there is a strong need for biomarker-supported differential diagnosis strategies for ND, able to supplement clinical and neurological ones. Although the etiology of Alzheimer’s disease (AD) is not very well-known, there is a common agreement that the accumulation of β-amyloid (Aβ) peptides in amyloid plaques is one of the hallmarks of the progression of the disease. Aβ peptides are formed by proteolytic cleavage of a transmembrane protein called amyloid precursor protein (APP).1-4 Cleavage at different sites is possible, and several Aβ peptides are found in measurable quantities in biological fluids of healthy individuals. For instance, five different Aβ peptides (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42) are regularly present at concentrations typically between 1 and 10 ng/mL in cerebrospinal fluid (CSF). Their detection in blood and urine,5-9 is much more difficult, since they appear at (1) Haass, C.; Schlossmacher, M. G.; Hung, A. Y.; Vlgo-Pelfrey, C.; Mellon, A.; Ostaszewski, B. L.; Lieberburg, I.; Koo, E. H.; Schenk, D. B.; Teplow, D. B.; Selkoe, D. J. Nature 1992, 353, 322–325. (2) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353–356. (3) Klein, W. L.; Krafft, G. A.; Finch, C. E. Trends Neurosci. 2001, 24, 219– 224. (4) Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.; Freed, R.; Liosatos, M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.; Viola, K. L.; Wals, P.; Zhang, C.; Finch, C. E.; Krafft, G. A.; Klein, W. L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6448–6453. (5) Wittke, S.; Mischak, H.; Walden, M.; Kloch, W.; Ra¨dler, T.; Wiedemann, K. Electrophoresis 2005, 1476–1487. (6) Hernandez-borges, J.; Neusu ¨ ss, C.; Cifuentes, A.; Pelzing, M. Electrophoresis 2005, 25, 2257–2281. (7) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (8) Seubert, P.; Vigo-Pelfrey, C.; Esch, F.; Lee, M.; Dovey, H.; Davis, D.; Sinha, S.; Schlossmacher, M. G.; Whaley, J.; Swindlehurst, C.; McCormack, R.; Wolfert, R.; Selkoe, D. J.; Lieberburg, I.; Schenk, D. Nature 1992, 359, 325–327. (9) Shoji, M.; Golde, T. E.; Ghiso, J.; Cheung, T. T.; Estus, S.; Shaffer, L. M.; Cai, X.; McKay, D. M.; Tintner, R.; Frangione, B.; Younkin, S. G. Science 1992, 258, 126–129.

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concentrations typically 10-100 times smaller. The ratios of concentrations of these different peptides are relatively stable in CSF of healthy individuals.10 In contrast, a selective reduction of Aβ1-42 in CSF from AD patients was reported in several studies, while the total level of Aβ peptides was found to be in the normal range.11 Conventional analytical methods such as enzyme linked immunosorbent assay (ELISA), Western blotting, and recently, capillary electrophoresis (CE) are used to measure the relative abundance of Aβ1-42 or aggregation process of Aβ in CSF.12-15 Recent advances demonstrated the potential of microfluidic capillary electrophoresis (MCE) as a reliable analytical method for the profiling of proteins and peptides in biological samples.16 As compared to conventional electrophoresis methods, MCE involves significant advantages, such as a low sample consumption and a strong potential for automation and integration.17,18 To our knowledge, though, MCE has not yet been applied for analysis of Aβ peptides in human CSF. One of the main challenges for the application of MCE to the analysis of Aβ peptides is adsorption onto the microchannel walls, notably in polymer chips, which are more hydrophobic than fused silica. Interactions of biomolecules with a microchannel wall tend to induce spurious band spreading and decrease analytical efficiency. This band spreading can arise from a “direct” adsorption of the analytes, typically leading to chromatographic effects, peak “tailing”, and in the worst cases, complete band suppression. Band spreading can also arise from an indirect, and often more difficult to characterize, flow profile disturbance associated with the adsorption of charged analytes or impurities on the channel’s wall, leading to noneven electroosmotic flow (EOF) and flow recirculation inside the microchannel.19 Therefore, different strategies have been developed to modify the surface of microchannels in polymeric microchips. These strategies can be divided into two main categories: covalent modification and what is called dynamic coating.20,21 In our laboratory, poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) was used as coating in polydimethylsiloxane (PDMS) chips for microchip isoelectric focusing (IEF) of protein samples.22 PDMA-AGE physically adsorbs to the surface of PDMS, and it effectively suppresses EOF in the PDMS microchannel. However, so far, its efficiency for reducing the adsorption of Aβ peptides to the surface of PDMS microchannels has not been investigated. (10) Wiltfang, J.; Esselmann, H.; Bibl, M.; Smirnov, A.; Otto, M.; Paul, S.; Schmidt, B.; Klafki, H. W.; Maler, M.; Dyrks, T.; Bienert, M.; Beyermann, M.; Ru ¨ ther, E.; Kornhuber, J. J. Neurochem. 2002, 81, 481–496. (11) Lewczuk, P.; Wiltfang, J. Proteomics 2008, 8, 1292–1301. (12) Varesio, E.; Rudaz, S.; Krause, K. H.; Veuthey, J. L. J. Chromatogr., A 2002, 974, 135–142. (13) Kato, M.; Kinoshita, H.; Enokita, M.; Hori, Y.; Hashimoto, T.; Iwatsubo, T.; Toyo’oka, T. Anal. Chem. 2007, 79, 4887–4891. (14) Sabella, S.; Quaglia, M.; Lanni, C.; Racchi, M.; Govoni, S.; Caccialanza, G.; Calligaro, A.; Bellotti, V.; De Lorenzi, E. Electrophoresis 2004, 25, 3186– 3194. (15) Verpillot, R.; Otto, M.; Klafki, H.; Taverna, M. J. Chromatogr., A 2008, 1214, 157–64. (16) Ohno, K.; Tachikawa, K.; Manz, A. Electrophoresis 2008, 29, 4443–4453. (17) Jakeway, S. C.; de Mello, A. J.; Russell, E. L.; Fresenius, J. Anal. Chem. 2000, 366, 525–539. (18) Chovan, T.; Guttman, A. Trends Biotechnol. 2002, 20, 116–122. (19) Hickey, O. A.; Harden, J. L.; Slater, G. W. Phys. Rev. Lett. 2009, 102, 108304. (20) Wu, D.; Qin, J.; Lin, B. J. Chromatogr., A 2008, 1184, 542–559. (21) Makamba, H.; Kim, J. H.; Lim, K.; Park, N.; Hahn, J. H. Electrophoresis 2003, 24, 3607–3619. (22) Shakalisava, Y.; Poitevin, M.; Viovy, J. L.; Descroix, S. J. Chromatogr., A 2009, 1216, 1030–1033.

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Apart from problems associated with sample adsorption and unstable EOF, achieving an acceptable resolution in MCE of Aβ peptides in PDMS chip is also quite challenging in terms of analytical selectivity. All of the five Aβ peptides of main interest mentioned above exhibit the same charge (theoretical isoelectric point of about 5), and they differ only by one or two amino acids in their carboxyl termini. Very weak differences in electrophoretic mobilites have made separation challenging in capillary zone electrophoresis (CZE) even with 50 cm long capillaries,15 and of course, the challenge will be worse in a microchannel only a few centimeters long, since in the simplest models for CZE, all other parameters being kept equal the resolution increases as the square root of separation length. Gel based MCE of peptides and proteins were shown to generally result in higher resolution than free zone electrophoresis.23 In capillary gel electrophoresis, linear polymers such as linear polyacrylamide,24 dextran,25 polyethylene oxide (PEO),26 pullulan,27 and hydroxylpropylcellulose28 were used for separation of proteins and peptides. Some of these polymers, such as polyacrylamide derivatives29 and cellulose derivatives have also been used in MCE.30,31 Here, we developed a gel based MCE method for analysis of fluorescently labeled Aβ peptides in the PDMS microchip. We studied different linear polymers and optimized chip treatment and separation conditions, in order to achieve the best resolution of five Aβ peptides. Finally, we validated this method toward the profiling of Aβ peptides in human CSF. Due to the complexity and variability of real CSF samples and to the low concentration of Aβ peptides, a sample preparation step prior to the MCE analysis was necessary. In order to pave the route toward a fully integrated, time effective, and user-friendly device, we also used for this step a microfluidic system. It is inspired from a system previously developed in our lab for enzymatic digestion of proteins.32,33 This system involves the self-assembly of biofunctionalized magnetic beads into a microcolumn in a microchannel comprised between two permanent magnets. Here, we coated the beads with anti-Aβ antibodies and used them to prepare an immunoaffinity column for the concentration and purification of Aβ samples. Finally, for control, we compared our results with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass analysis on the same samples. (23) Mohamadi, M. R.; Kaji, N.; Tokeshi, M.; Baba, Y. Anal. Chem. 2008, 80, 312–316. (24) Chiari, M.; Nesi, M.; Fazio, M.; Righetti, P. G. Electrophoresis 1992, 13, 690–697. (25) Ganzler, K.; Greve, K. S.; Cohen, A. S.; Karger, B. L. Anal. Chem. 1992, 64, 2665–2671. (26) Tseng, W. L.; Chang, H. T. J. Chromatogr., A 2001, 924, 93–101. (27) Hu, S.; Zhang, L.; Cook, L. M.; Dovichi, N. J. Electrophoresis 2001, 22, 3677–3682. (28) Hu, S.; Zhang, Z.; Cook, L. M.; Carpenter, E. J.; Dovichi, N. J. J. Chromatogr., A 2000, 894, 291–296. (29) Bousse, L.; Mouradian, S.; Minalla, A.; Yee, H.; Williams, K.; Dubrow, R. Anal. Chem. 2001, 73, 1207–1212. (30) Mohamadi, M. R.; Mahmoudian, L.; Kaji, N.; Tokeshi, M.; Baba, Y. Electrophoresis 2007, 28, 830–836. (31) Mohamadi, M. R.; Kaji, N.; Tokeshi, M.; Baba, Y. Anal. Chem. 2007, 79, 3667–3672. (32) Le Nel, A.; Minc, N.; Smadja, C.; Slovakova, M.; Bilkova, Z.; Peyrin, J. M.; Viovy, J. L.; Taverna, M. Lab Chip 2008, 8, 294–301. (33) Slovakova, M.; Minc, N.; Bilkova, Z.; Smadja, C.; Faigle, W.; Futterer, C.; Tavernac, M.; Viovy, J. L. Lab Chip 2005, 5, 935–942.

Figure 1. (A) Sample preparation unit: magnetic beads coated with anti-Aβ are self-organized in the microfluidic channel of a microfluidic chip made of PDMS and comprises two permanent magnets. Eluted samples are collected in an Eppendorf tube. (B) Principle of analysis, made in parallel by MALDI-TOF and MCE. MCE is performed in a cross channel pinch injection microchannel layout, made of PDMS. Surface treatment of a PDMS chip involves two sequential layers. The first layer is noncovalent physical coating by PDMA-AGE, and the second layer is dynamic coating by adding methylcellulose and Tween-20 in the electrophoresis buffers.

EXPERIMENTAL SECTION Reagents, Polymer, and Sample Preparation. Aβ peptides (1-37, 1-38, 1-39, 1-40, and 1-42) were purchased from AnaSpec (Fremont, CA). Fluoprobe-488 NHS ester was purchased from Interchim (Montlucon, France). Methylcellulose (MC-400) was purchased from Sigma Chemical Co. (St. Louis, MO). The methylcellulose solutions were prepared according to the manufacturer’s suggestions. Linear polyacrylamide (LPA; MW 600 0001 000 000) 10% solution in water was purchased from Polysciences Inc. (Warrington, PA). Microchip Preparation. Microchips were fabricated by rapid prototyping and PDMS technology,34 starting with a master composed of a positive relief of SU-8 resin on a silicon wafer (Neyco, France) made by soft lithography. A PDMS replica of the master was formed. The PDMS was then peeled away, and holes were pierced for tubing connection and for the reservoirs. The replica was permanently sealed with a PDMS-coated glass slide. Bonding was enhanced and made irreversible by oxidizing both the replica and the cover in a plasma discharge for 1 min prior to bonding. Two types of chips were used in this research (Figure 1): one for immunocapture and one for MCE. For immunocapture chips, a Plexiglas (polymethylmethacrylate) mold containing plastic relief which precisely define the size and location of the magnets was used. The channel cross section in the immunocapture chip was 1 mm (width) by 200 µm (depth), and the channel length was 2 cm. Silicone tubing was then added at the inlet and the outlet of the channel to inject and collect different solutions. In the MCE chip, the channel cross section was 100 µm (width) by 50 µm (depth), and the effective separation length was 35 mm. The distances from the intersection of microchannels to the buffer reservoir, buffer waste reservoir, the sample reservoir, and sample waste reservoir were 5, 37.5, 5, and 5 mm, respectively. Labeling of Aβ Peptides. Aβ peptides were dissolved in an alkaline buffer and labeled with Fluoprobe-488 NHS prepared in DMSO at concentration of 10 mg/mL (according to a protocol developed previously by Verpillot et al., manuscript in preparation). Typically, a 10-fold excess of the fluorescent dye was used for labeling. The mixture was then incubated at 4 °C for 1 h in the dark. (34) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 550– 575.

Preparation of CSF Samples. The CSF samples analyzed in this study were obtained from patients attending the outpatient memory clinic in Ulm. Collection and analysis of the CSF sample were approved by the Ethics Committee in Ulm. CSF was taken, aliquoted within 2 h, and stored at -80 °C. CSF was directly used for the labeling reaction by adding 10 µL of Fluoprobe NHS-488 (10 mg/mL in DMSO) to 50 µL or 250 µL of human CSF. The mixture was incubated at 4 °C for 1 h in the dark. The labeled CSF was then used for immunocapture of Aβ peptides. In some experiments, fluorescent labeling was performed after immunocapture and on the eluted peptides (see Results and Discussion); in this case for peak identification and to reduce the intensity of the peaks for the fluorescent dye, we used centrifugal filter units 3K (Millipore, Billerica, MA). Affinity Immunocapture of Labeled Aβ Peptides in Microchip and in Batch. For immunocapture of Aβ peptides, we used a semiautomated immunocapture method in a microchannel (Figure 1A). Two rare earth permanent magnets (Neodynium Iron Boron, Hangzhou, China) were inserted into the cut-outs of the PDMS block. Both magnets have dimensions of 4 mm × 4 mm ×16 mm, with a magnetic remanence of 1.3 T at the pole and a polarization in the longest dimension. The magnets point in the same direction and make an angle of 20° with the channel. Magnetic microparticles (SiMAG-Hydrazide 1 µm from Chemicell, Germany) were coated with monoclonal antibody anti-Aβ 6E10 (Covance, Emeryville, CA). Anti-Aβ antibodies (100 µg) were coupled to 1 mg of magnetic microparticles, following the procedure suggested by the supplier (Chemicell, Germany). The binding specificity to all Aβ isoforms and a close to zero level of nonspecific adsorption were confirmed by conventional gel electrophoresis and Western blotting. One milligram of the antibodycoated microparticles was suspended in PBS-T buffer. The suspension was then injected into the microchannel with a flow rate of 2 mL per hour. When approaching the magnet zone, the beads self-organize in columns oriented in the direction of the flow. They remain trapped between the magnets, owing to the magnetic field gradient, and accumulate to form a dense plug. The plug was then rinsed with PBS buffer at the same flow rate, and the sample solution was finally injected. After several washing steps, the captured Aβ peptides were eluted by 0.16% NH4OH. For immunocapture of CSF, the CSF sample was mixed with the same volume of RIPA buffer (50 mM Tris-HCl, pH ) 7.2; 150 mM Analytical Chemistry, Vol. 82, No. 18, September 15, 2010

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NaCl; 1% Triton X-100; 0.1% SDS; 1 mM Na3VO4; 1 mM PMSF; 1 mM EDTA; 1 mM protease inhibitor). RIPA buffer was also used for the first washing step. For the second washing step and elution, we used the same buffer as for standard peptides. The eluted Aβ peptides were divided into two aliquots and reconcentrated in speedvac. One aliquot of the eluted sample was redissolved in 15 µL of borate buffer (40 mM, pH 10.5) and was used for MCE analysis. The second part was kept for MALDI-TOF mass analysis. Microchip Electrophoresis: Surface Coating of PDMS Channel and EOF Measurements. The primary surface coating was achieved by introducing a solution of 0.2% of a homemade copolymer PDMA-AGE in ddH2O into the microchannels of a plasma treated PDMS microchip.22 The chip was kept in the dark for at least 30 min, and the excess polymer was rinsed out by extensive ddH2O washing. After coating the PDMS chip with PDMA-AGE, methylcellulose at various concentrations (at least 0.2%) plus 0.01% Tween-20 was added to the electrophoresis buffers. EOF was measured using the current monitoring method.30,31 Microchip Electrophoresis: Separation. A HVS448 3000 V power supply (LabSmith, Livermore, CA) controlled by a PC was used for MCE. Sample was injected to the electrophoresis channel by a pinched injection method. The applied voltages for each electropherogram are mentioned in the related figure caption. An inverted fluorescence microscope, Olympus IX71, equipped with a 10× 0.3 NA objective lens (Olympus, Tokyo, Japan) was used to detect in real time fluorescently labeled peptides. Illumination was done by a 100 W mercury arc lamp. A Nikon digital DS-Qi1 camera (Nikon, Tokyo, Japan) was used to capture images of the detection zone. Still images were recorded at a frequency of 10 images per second; intensity from the selected detection zone was integrated using the image processing software (NIS Elements by Nikon), and the intensity corresponding to each frame was recorded in an electropherogram for each separation. MALDI-TOF Mass Spectrometry of Immunoprecipitated Aβ Peptides. Samples were redissolved in 60% acetonitrile/0.1% trifluoroacetic acid. An aliquot of 0.5 µL was mixed with 0.5 µL of saturated a-cyano-hydroxycinnamic acid, 50% acetonotrile, and 0.1% trifluoroacetic acid and was spotted onto a MALDI plate and allowed to dry. All spectra were acquired with a 4800 plus MALDITof/Tof mass spectrometer (Applied Biosystems, Foster City, CA). RESULTS AND DISCUSSION Resolving Aβ peptides with very similar properties in a microchannel much shorter than CE capillaries or conventional gels was a major challenge of this project, and we first optimized this separation. This involved first achieving an efficient surface coating able to control the EOF and to suppress sample adsorption into the microchannel and second achieving the best peak separation possible. Physical and Dynamic Coating of PDMS Surface. MCE in a bare PDMS chip failed due to high electroosmotic flow that hindered sample injection into the electrophoresis channel. EOF in uncoated PDMS was 4.5 × 10-4 cm2 · v-1 · s-1 which was higher than the average mobility of Aβ peptides (1.5 × 10-4 cm2 · v-1 · s-1), and therefore, MCE of Aβ peptides in an uncoated PDMS chip was not possible. To suppress the EOF, we used a solution of a copolymer (PDMA-AGE) in ddH2O as 7614

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physical coating. After coating by PDMA-AGE, EOF decreased by 2 orders of magnitude (1 × 10-6 cm2 · v-1 · s-1). However, following MCE of fluorescently labeled Aβ peptides in microchannels coated with PDMA-AGE, we found considerable fluorescent signals on the surface of the microchannel, the signature of strong sample adsorption onto the surface of microchannel. By adding 0.2% methylcellulose and 0.01% Tween-20 to our electrophoresis buffer, the remaining fluorescent signal on the surface of the microchannel was negligible. We had previously shown that methylcellulose-Tween performs efficient dynamic coating on plastic (PMMA) microchips,30 and we showed here that this coating is also applicable on the PDMS chip precoated with PDMA-AGE. In contrast with PMMA, however, methylcellulose-Tween alone in the PDMS channel did not suppress the EOF. Therefore, the best performance for the separation of peptides was achieved when a double coating (PDMA-AGE and methylcellulose-Tween) was used, as shown in Figure 1B. MCE of Aβ Peptides in LPA. For optimization of MCE, we started our experiments by separation of two fluorescently labeled Aβ peptides (Aβ1-37 and Aβ1-42). As separation media, first, we used different concentrations of LPA. We achieved the separation of two separated peaks Aβ1-37 and Aβ1-42 in 5% LPA. However, we found that, after a few tests in the same chip, EOF in the microchannel increased and the sample introduction to the electrophoresis channel failed. EOF analysis showed that, when we performed five successive analyses in the same chip using LPA, the EOF increased by 1 order of magnitude (5.12 × 10-5 cm2 · v-1 · s-1). We concluded that LPA is not compatible with the PDMA-AGE coating on the PDMS surface. MCE of Aβ Peptides in Methylcellulose Solutions. As media for separation, we also used several concentrations of methylcellulose in borate buffer. The results showed that, by increasing the concentration of methylcellulose, the resolution of two peptides was improved. In 0.2% methylcellulose, for example, two peaks for Aβ1-37 and Aβ1-42 were not resolved, but using 2% methylcellulose, we achieved the full resolution of the two peptides. Higher concentrations of methylcellulose had too high viscosity to allow for reproducible introduction into PDMS microchannels. Therefore, subsequent experiments were performed using 2% methylcellulose in borate buffer. We also optimized buffer pH and electrophoresis voltages, and we showed that Aβ1-37 and Aβ1-42 were better separated in 40 mM borate buffer, pH ) 9.5 (Figure 2A). The stability of PDMA-AGE coating in the presence of methylcellulose solution was excellent, and we obtained a good reproducibility of peak areas and migration times over repeated separations of a mixture of Aβ1-37 and Aβ1-42 (RSD for run to run analysis in the same chip was less than 1% (n ) 5 data not shown)). EOF analysis showed that using methylcellulose as electrophoresis media after several tests of EOF remained negligible (1 × 10-6 cm2 · v-1 · s-1). Validation of Profiling of Aβ Peptides by MCE. As mentioned in the introduction, the absolute amount of Aβ peptides vary quite significantly between different healthy or diseased individuals, but a reduced relative concentration of Aβ1-42 as compared to other Aβ peptides is a clinically relevant parameter for the diagnosis of AD. Therefore, to validate our MCE method,

Figure 2. Electropherograms of a mixture of Aβ1-37 and Aβ1-42 with different concentration ratios. The peptides were labeled with NHSFluoprobe-488. (A) The electropherograms are zoomed for the area where the peaks for Aβ1-37 and Aβ1-42 appear. (B) Shows the correlation of the ratios of the peak areas and the concentrations for Aβ1-37 and Aβ1-42 in (A). All separations were performed in 2% methylcellulose in 40 mM borate buffer, pH ) 9.5. To inject the sample, 550 V was applied for 45 s. Pull back and electrophoresis voltages were 250 V and 950 V, respectively.

Figure 4. Electropherogram from the separation of a mixture of five Aβ peptides (Aβ1-37(1), Aβ1-38 (2), Aβ1-39 (3), Aβ1-40 (4), and Aβ142 (5)). Each peptide was labeled with Fluoprobe-488 and purified using the immunocapture chip. MCE conditions were the same as in Figure 2.

Figure 3. (A) Shows the electropherograms of Fluoprobe-488 labeled standards Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42. Similar concentrations of each peptide (1 µg/mL) were labeled and purified using the immunocapture chip in Figure 1A. P is indicating the peaks related with the labeled peptides, and P* are different modifications of the main peak. F and F′ indicate the peaks related with the fluorescence dye. MCE conditions were the same as in Figure 2. (B) Shows mass spectra of individual Aβ peptides as it is indicated in (A). 1F, 2F, and 3F in (B) correspond to the number of fluorescent dye per peptides.

we prepared and analyzed mixtures of various ratios of Aβ1-37 and Aβ1-42 (Figure 2). We found a linear correlation between concentration ratios of the peptides and ratios of both peaks heights and peak areas (R2 were 0.99 and 0.98 respectively; Figure 2B). Immunocapture Followed by MCE of Five Aβ Peptides. We used an on-chip immunocapture system to isolate labeled Aβ peptides from the excess of fluorescent dye remaining after the labeling step. The electropherograms in this case mostly includes the peaks for labeled Aβ peptides. In Figure 3, electropherograms

from the analysis of Aβ peptides individually labeled and purified and results of mass analysis of each labeled peptide are presented. As compared to Figure 2A, the peaks associated with free fluorescent dyes are significantly decreased. In the electropherograms in Figure 3A, we can see the peaks for each Aβ peptide and some additional minor peaks, labeled P*. With our MCE method, it was not possible to characterize these minor peaks (P*); as a first interpretation, they might correspond to variations in the label numbers per each peptide, since MS spectra show minor peaks corresponding to such species. However, an additional argument suggests that these peaks are indeed rather due to peptide oligomerization. We noted that the amplitude of these P* peaks tend to increase upon storage of the peptide solution after preparation. Note that this problem is specific for purified peptide solutions, since the state of oligomereization of Aβ peptides in CSF is presumed to be stable. Also, the mass spectra in Figure 3B show that under our labeling conditions the peptides are mainly labeled with two fluorescent dyes. In Figure 4, finally, a separation of a mixture of five Aβ peptides is presented (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42), showing that all peptides except Aβ1-38 from Aβ139 are resolved. Although baseline resolution of all peaks in MCE was not complete, the relative abundance of Aβ-42 can be determined using standard peak analysis methods, and this should Analytical Chemistry, Vol. 82, No. 18, September 15, 2010

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Figure 5. (A) Electropherogram of labeled CSF. CSF (250 µL) was labeled with Fluoprobe-488, and the Aβ peptides in the labeled CSF were isolated using magnetic beads coated with anti-Aβ in the immunocapture chip. An electropherogram of standard Aβ1-40 labeled with Fluoprobe488 is superimposed to identify the peak from Aβ1-40 in CSF. Aβ* corresponds to the peak for aggregated or modified peptides. (B) Electropherogram of Aβ peptides immunocaptured from 250 µL of CSF which were labeled after immunocapture. An electropherogram (dashed electropherogram) of a blank control showing the peaks related with the fluorescent dye is superimposed in this electropherogram. The separation window of Aβ1-40 and Aβ1-42 is out of the zone for strong peaks related with the fluorescent dye. MCE was performed in 2% methylcellulose, and separation conditions were as in Figure 2.

be sufficient for diagnostic purposes. Indeed, currently, more cumbersome methods like sodium dodecyl sulfate (SDS)-urea polyacrylamide gel electrophoresis (PAGE) allow resolving of the five peptides (Supporting Information, Figure S.1), but from the clinical analysis, only the relative abundance of Aβ-42 is considered a valid biomarker of neurodegenerative disease.35 Finally, for the future, it is worth to note that, unlike the SDS-urea PAGE, the MCE method presented here works in a native peptide state and may prove to be able to monitor the aggregation state of Aβ peptides. Recently, electrophoresic separation in the native state was performed for studying oligomerization of Aβ peptides.36 Oligomerization of Aβ peptides is not yet a validated clinical biomarker, but it currently raises intense interest at the clinical research level and may prove to be useful to support the diagnosis in the future.36 MCE of Aβ Peptides in Human CSF. Finally, we tested our method against CSF samples from AD and nonAD patients. The first problem encountered there was the efficiency of labeling, combined with the low abundance of Aβ peptides as compared to the large amount of other proteins and peptides in CSF. The latter forced us to use higher concentrations of fluorescent dye (see the Experimental Section for details). MCE and MALDI-TOF were performed after immunocapture and isolation of Aβ peptides from labeled CSF. As it is shown in Figure 5A, we could only detect Aβ1-40 which is the most abundant Aβ peptide in human CSF. To improve the sensitivity of detection, we adopted an alternative labeling method, in which immunocapture was done on unlabeled CSF and the Aβ peptides were labeled after elution. This way, we were able to detect at least two peaks related with Aβ peptides in CSF (Figure 5B). By adding internal standards and filtration of the fluorescence dye, we could identify the peaks for Aβ1-40 and Aβ1-42. Other Aβ peptides in this method comigrated with the peaks from fluorescent dyes and could not be seen in the electropherogram. The results of MALDI-TOF mass

analysis also showed that under the same labeling conditions only Aβ1-38 and Aβ1-40 could be detected in the CSF. Aβ1-42, which is the most hydrophobic Aβ peptide, generally produces a weaker signal in MS.37 We tested the CSF samples from four non-AD and three patients with AD. One representative pattern of all analyzed samples showing Aβ1-40 and Aβ1-42 can be seen in Figure 5B. These two peaks repeatedly appeared in the samples and were not seen in the negative control (blank run with the peaks for the fluorescent dye). However, a quantitative differentiation of the ratio of Aβ1-40 to Aβ1-42 between AD and nonAD patients was not achieved. This could be due to high interference of the peaks for the fluorescence dye and limited number of the samples.

(35) Blennow, K. Expert Rev. Mol. Diagn. 2005, 5, 661–672. (36) Wiberg, H.; Ek, P.; Pettersson F. E.; Lannfelt, L.; Emmer, A.; Roeraade, J. Anal. Bioanal. Chem. 2010, 397, 2357–2366.

(37) Portelius, E.; Tran, A. J.; Andreasson, U.; Persson, R.; Brinkmalm, G.; Zetterberg, H.; Blennow, K.; Westman-Brinkmalm, A. J. Proteome Res. 2007, 6, 4433–4439.

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CONCLUSION In summary, we developed a strategy for profiling Aβ peptides in CSF, in which both sample enrichment and electrophoretic separation can be performed on chip. On model samples, we demonstrated for the first time reproducible separation of differently truncated Aβ peptides and a quantification of the relative abundance of Aβ1-42, a biomarker of considerable clinical importance. This was obtained thanks to a new coating strategy for PDMS chips, combining a first layer of PDMA-AGE and a second dynamic coating by methyl cellulose. With real CSF from patients, we also developed a strategy allowing the detection and separation of some peptides, but these proof-of-concept results still call for improvements, notably regarding sensitivity, before this approach can be used routinely. The sensitivity of detection in this method is not yet sufficient for the detection of Aβ peptides in CSF without preconcentration. We believe, however, that the current work has provided new clues toward this direction. In particular, one of our future goals is to develop a fully integrated chip with online preconcentration in order to reach the sensitivity necessary for detection of Aβ peptides in CSF.

ACKNOWLEDGMENT This work was supported by European Grant LSHB CT 06 037953 (NeuroTAS project). The authors would like to thank Wolfgang Faigle from the Mass Spectrometry Laboratory in the Curie Institute for performing mass analysis of our samples and also Hans-Wolfgang Klafki from University of Duisburg-Essen for his valuable comments and discussion on SDS-Urea PAGE and western blotting analysis of Aβ peptides.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review May 21, 2010. Accepted August 2, 2010. AC101337N

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