Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by

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Microfluidic Isoelectric Focusing of Amyloid Beta Peptides followed by Micropillar-MALDI-Mass Spectrometry Saara Mikkonen, Johan Jacksén, Johan Roeraade, Wolfgang Thormann, and Åsa Emmer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02324 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Analytical Chemistry

Microfluidic Isoelectric Focusing of Amyloid Beta Peptides followed by Micropillar-MALDI-Mass Spectrometry Saara Mikkonen,*† Johan Jacksén,† Johan Roeraade,† Wolfgang Thormann‡ and Åsa Emmer† †

Department of Chemistry, Div. of Applied Physical Chemistry - Analytical Chemistry, KTH Royal Institute of Technology, Stockholm, Sweden ‡ Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland ABSTRACT: A novel method for preconcentration and purification of the Alzheimer’s disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-MALDI-TOF-MS is presented. A semi-open chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer) and arginine, which provided a step-wise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder affecting around 35 million people worldwide, and due to population ageing, this number is expected to grow.1 Today no cure or efficient treatment exists. Nevertheless, it has long been realized that early and differential diagnosis is important, and there has been an intensive search for suitable biomarkers. Several studies have associated elevated levels of total and phosphorylated tau protein and reduced amyloid beta (Aβ) 1-42 peptide in cerebrospinal fluid (CSF)2-4 with AD (for a meta-analysis see the recently published review by Olsson et al.5). Aβ peptides are generated by β- and γ-secretase cleavage of the amyloid precursor protein (APP) (resulting in a variety of truncated isoforms) and can form neurotoxic aggregates, which eventually accumulate as amyloid plaques, mainly consisting of Aβ 1-42.6,7 In CSF of AD patients Aβ 1-42 levels are reported to be in the pg/mL range and are higher compared to those in plasma.8 A selective reduction of Aβ 1-42 in CSF, often measured as the ratio between Aβ 1-42 and Aβ 1-40, is used as AD biomarker.8,9 Typically these peptides are detected using immunoassays, e.g. enzyme-linked immuno-sorbent assays (ELISAs), including optical detection.1 Although this approach offers high sensitivity, possible non-specific interactions or cross reactions with the employed antibodies may lead to incorrect results. Moreover, pathologically relevant information on potentially important posttranslational modifications is not obtained. Mass spectrometry (MS) is able to provide a molecular identity and also facilitates simultaneous detection of a multitude of peptides. This is important since Aβ peptides other than those for which there are commercially available ELISAs (i.e. 1-38, 1-40 and 1-42) have

attracted clinical research interest.1,9,10 The ability of simultaneous detection and unequivocal identification of several Aβ isoforms is also essential for analysis of Aβ peptides in plasma. The pattern of Aβ peptides in plasma has been suggested to be of use in clinical trials as a measure of response to treatment,8,11 or as a prognostic biomarker when measured repeatedly in the same individual.12 A correlation between Aβ levels in CSF and plasma has not been established.5 Recently, however, weak but statistically significant correlations were reported.13 Furthermore, Kaneko et al. detected novel Aβ isoforms in plasma by MALDI-TOF-MS, opening the possibility of finding plasma based AD biomarkers.14,15 This would be highly desirable due to the ease of plasma sampling compared to invasive and painful lumbar puncture required for CSF collection. MS detection of Aβ peptides in CSF and plasma is demanding because of their low abundance and a preconcentration and cleanup step is thus required. The methods, which have been employed, include immunoprecipitation (IP) and solid phase extraction (SPE)16. SPE is an inexpensive and simple technique, but suffers from a severe lack of selectivity. In comparison, antibody-based approaches are superior. IP and in particular the combination of IP and MS has shown the most promising results and has been extensively used for analysis of Aβ peptides after the signaling paper of Portelius et al.17 in which the detection of Aβ signatures in CSF using IP-MALDI-TOF-MS is described. In addition to IP enrichment, the MALDI-MS sensitivity can be increased by confinement of the sample into a small area.18 We have previously developed silicon chips with elevated micro structures as targets for MALDI-TOF-MS.19,20 When using these micropillar targets for detection of Aβ in CSF and plasma samples

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it was, however, found that the IP eluate contained unknown contaminants. In this work, we present an approach for obtaining a further purification of the IP eluate using isoelectric focusing (IEF) in a 75 nL microchannel prior to MALDI-TOF-MS. In other studies, IEF has been used for separation of Aβ peptides in CSF employing 2-D gel and capillary immunoassay formats.21,22 Herein, computer simulation was used to design a step-wise pH gradient tailored for preconcentration and purification of Aβ peptides in human CSF and plasma. The pH gradient was created with a few simple, chemically well-defined carrier components in a similar way to what was previously employed for the enrichment of model peptides in microchannels.23 The experimental setup consisted of an open microchannel system covered with a liquid layer of fluorocarbon24 to avoid premature evaporation while providing access to the sample. Thereby, straight-forward sampling of the focused Aβ peptides for subsequent micropillarMALDI-TOF-MS was enabled. EXPERIMENTAL SECTION Simulations. The simulation software used, GENTRANS, has been described previously.25-27 Briefly, it is a one-dimensional simulator based on the principles of electroneutrality and conservation of mass and charge. To compute component fluxes, contributions of electromigration, diffusion and convection are included. For simulations, a 1 cm column was divided into 1000 segments (∆x=10 µm). Uniform initial distributions of all components along the channel and absence of any fluid flow were assumed together with IEF boundary conditions at the column ends (permeable only to H+ and OH-) and a constant 74.0 V was applied. Other input parameters used are listed in Tables S-1 and S-2 in the Supporting Information. For most small molecular mass components, mobility and pKa values were taken from the SIMUL5 database (available at http://web.natur.cuni.cz/~gas/). The pKa values for the dipeptide Asp-His were taken from the literature.28 For the peptides and proteins theoretical titration curves and diffusion coefficients were calculated based on amino acid composition and molecular mass, respectively, as described earlier.23 The amino acid sequence of the anti-β-amyloid 1-16 antibody (6E10) was obtained from the literature.29 Aβ peptide sample preparation. Standards of Aβ 1-38 and 140 were prepared according to the manufacturers instructions (rPeptide, Bogart, GA, USA). CSF samples from patients diagnosed with AD were kindly provided by Sanna-Kaisa Herukka at the Institute of Clinical Medicine/Neurology, University of Eastern Finland, and the plasma samples were from a healthy volunteer who gave her consent. A total of 18 samples were available for method development and analysis. Before IP, CSF and plasma were stored at -80 ºC. IP of 800 µL CSF/plasma was performed using magnetic Dynabeads M-280 Sheep Anti Mouse IgG covalently coated with 6E10 as capture antibody (obtained from Ute Haussmann, LVRKlinikum Essen, Department of Psychiatry and Psychotherapy, University of Duisburg-Essen). A previously described IP protocol30 was employed with modified washing steps (three times with PBS (Biochrom, Cambridge, UK) /0.1% BSA (Sigma Aldrich, Stockholm, Sweden), two times with 50 mM NH4HCO3 (AppliChem, Darmstadt, Germany) and one time with water). All water used was deionized water by a Milli-Q Synergy 185 system (Millipore, Bedford, MA, USA). The elution of the peptides was performed at 95ºC22 with 50 µL water during 5 min. This solution was vacuum centrifuged to a final volume of approx. 1.5 µL (which will be referred to as IP eluate in the text to follow). In

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some experiments, the eluate was vacuum centrifuged to dryness and redissolved in IEF buffer. Open channel IEF combined with micropillar-MALDI-MS. An IEF buffer consisting of 5 mM each of glutamic acid (Glu, VWR, Stockholm, Sweden), aspartyl-histidine (Asp-His, AnaSpec, Liege, Belgium), cycloserine (cSer, Sigma Aldrich), arginine (Arg, VWR) and 0.05 or 0.1% trifluoroacetic acid (TFA, Sigma Aldrich) was used. The open microchannels, fabricated on silicon microchips had the following dimensions: length: 1 cm, width: 150 µm and depth: 50 µm, yielding a channel volume of 75 nL. The chip fabrication procedure has been described before.31 Aβ peptide samples (standard solutions or IP eluate) were mixed with the IEF buffer and the mixture was supplied to the open microchannel using a pipette. In other experiments the IP eluate was directly loaded into the microchannel through a fused-silica capillary (id/od 15/150 µm) (CM Scientific, Silsden, UK) and IEF buffer was added thereafter. In the latter case, sample volumes larger than the 75 nL channel volume were loaded through evaporation of water from the open system during sample application as a means of further concentrating the sample. Some samples were divided into two channels to assure the methods repeatability. A schematic representation of the instrumental setup is depicted in Figure 1. The microchannel and micropillar chips were placed in a glass cuvette on a robotic xyz-translational stage equipped with two CCD-cameras20,23 and covered by an inert fluorocarbon liquid (FC-770, 3M, St. Paul, MN, USA) lid.32 A constant voltage of 74 V was applied across the channel length for 10-15 min (Figure 1A). Further details of the instrumental setup for IEF have been described elsewhere.23 After IEF, the tip of an approx. 3 cm long fused-silica capillary (id/od 10/150 or 15/150 µm), etched to a cone-shape using hydrofluoric acid (HF, 50% VLSI Selectipur, Merck, Darmstadt, Germany), was positioned in the channel at the location of the focused zone (Figure 1B). A pulse of vacuum suction was applied to sample 2.5 or 5 nL containing the focused peptides, for subsequent deposition onto elevated pillars (height 80 µm)19 with diameters of 100 or 200 µm, respectively, by applying a pulse of compressed air (Figure 1C). The fluorocarbon layer was then removed by suction using a pipette and MALDI matrix (10 mg/mL 2,5-dihydroxybenzoic acid (DHB, Bruker Daltonik, Bremen, Germany) in 9/1 0.1% TFA/acetonitrile (Merck)) was deposited onto the pillars through a fused-silica capillary (id/od 15/150 µm) until a clear crystallization could be visually observed. For the CSF and plasma samples, recrystallization was performed by adding a few nanoliters of 1% TFA. The chip was mounted onto a custom-made stainless steel plate (Mikroverktyg AB, Södertälje, Sweden), and MALDI-MS (ultrafleXtreme, Bruker Daltonik) was performed using a reflectron positive method. Data evaluation was performed with FlexAnalysis software (Bruker Daltonik).

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Figure 1. Schematic instrumental setup for IEF, sampling from the open microchannel and sample deposition onto micropillar MALDI target. Inset: photograph of the deposition. MALDI-MS from open channels. We have previously Table 1. Isoelectric points and molecular masses of different demonstrated that the open silicon microchannel can be used as a C-terminal amyloid beta peptides. MALDI target.31,33 After focusing of the peptides, the fluorocarbon was removed and the water was allowed to evaporate to dryness while maintaining the voltage across the channel. Thereafter, MALDI matrix (45 mg/mL DHB in 1/1 0.1% TFA/ethanol (Sigma Aldrich) with 0.25 or 0.5 µM neurotensin (Sigma Aldrich) as internal standard) was applied using electrospray matrix deposition (as described earlier33). The chip was mounted in a TLCMALDI adapter (Bruker Daltonik) and the S/N of the Aβ peptides, relative to the internal standard, was measured along the channel length. RESULTS AND DISCUSSION Simulations. Computer simulation is a powerful tool in predicting the behavior of electrophoretic systems. To simulate IEF in complex mixtures of commercial carrier ampholytes, many approximations regarding their electrochemical properties must be made, which limits the possibility of an accurate prediction of the location of the isoelectrically focused species.34 By using simpler and chemically well-defined carrier components, a better correlation between simulations and experiments can be expected, and by proper selection of these components, pH gradients tailored for specific applications can be designed.23 Most C-terminal Aβ peptide isoforms starting with aspartic acid-1, including Aβ 1-38, Aβ 1-40 and Aβ 1-42, share a pI value of 5.3 (see Table 1). The aim was thus to create a pH step around this value; sufficiently narrow to minimize the risk of co-focusing of peptide or protein contaminants, but wide enough to allow for possible differences between theoretical and actual pIs. Theoretical titration curves calculated from pKa values for the amino acid residues obtained from the database of SIMUL5 correlated well with those generated by the ExPASy ProtParam tool (http://web.expasy.org/protparam) (Table 1), and were used as input in the simulations (Table S-2). The theoretical pI values are also in agreement with those measured experimentally with capillary IEF immunoassay.22 A carrier ampholyte must be able to carry both current and pH in its isoelectric state. As stated already in 1962 by Svensson, suitable components should be isoelectric between two closely spaced pKa values, i.e. they have a low pI-pKa1 or ∆pKa.35

1: pI values estimated graphically from theoretical titration curves calculated using pKa values for amino acid residues from the SIMUL5 database. 2: pI values generated by ExPASys ProtParam tool. Amino acid sequence of Aβ 1-42: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV VIA Asp-His and cSer, with pI values 4.9 and 5.9, respectively, fall within this category, and were used together with Glu and Arg (Table S-1). Similar systems were previously used e.g. to explore the focusing mechanism in IEF36,37 and for protein fractionation in a suspended drop38. Upon voltage application, these components migrate to create a step-wise pH gradient (Figure 2A and 2C).

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Figure 2. IEF of 1 µM Aβ 1-38 and 1-40 and 0.1 µM sAPPα and 6E10 in a mixture composed of Glu, Asp-His, cSer and Arg (5 mM each) and commencing with a uniform component distribution. The plots show computer simulated (A) ampholyte (B) peptide, (C) pH and (D) conductivity distributions after 1, 2, 5 and 10 min (from top to bottom, respectively) predicted for application of a constant 74.0 V. The initial pH and conductivity were 5.69 and 27.3 mS/m, respectively. The anode is on the left. The formed zones are also demarcated with changes in conductivthat the acid migrates towards the anode, forcing the other components in the channel to a position closer to the cathode. The ity (Figure 2D). In this system, the Aβ peptides will focus in the pH step in the boundary between the Asp-His and cSer zones. amount of TFA in the sample influences the magnitude of this position shift. Formation of the anticipated pH step for Aβ focusThis is demonstrated in simulations with the Aβ 1-38 and Aβ 1-40 peptides (Figure 2B). ing is, however, predicted to be unaffected by the presence of TFA. Initial work on micropillar-MALDI-MS of Aβ peptide samples revealed that the IP eluate contained unknown contaminants. The dynamics of IEF are composed of a fast separation phase and a much slower stabilizing phase eventually leading to a After deposition onto a micropillar and evaporation of the water, a viscous consistency could be observed for some samples (for steady-state configuration.40 Due to the slow migration of the photographs see Fig. S-1 in the Supporting Information). This acid, steady-state has not been reached at the 10 min time point prevented crystallization of the MALDI matrix, and consequently shown in Figure 3, although full separation of the peptides and no Aβ peptides could be detected (data not shown). Generally, no proteins has been achieved. For sampling from a specific channel more than 5 nL of the IP eluate could be deposited without risking position during the stabilizing phase, i.e. before steady-state has loss of signal. been reached, it is important to sample at the same time to obtain comparable results. In a system with 0.05% TFA (6.4 mM initial The monoclonal antibody 6E10 is one of the most common anconcentration), the Aβ peptides migrate to a position approx. 6 tibodies used to capture Aβ peptides and is directed towards the 1 mm from the anode after application of 74 V/cm for 10 min (Figepitope 4-9. Thus, it also recognizes the soluble amino-terminal ure 3A), whereas an initial TFA concentration of 0.1% (12.9 mM) APP fragment alpha (sAPPα), produced in the non-amyloidogenic results in an Aβ peptide position close to 7 mm from the anode pathway of APP processing.8 sAPPα might therefore be present in (Figure 3B). For the used configurations, steady-state was preimmunoprecipitated Aβ samples. Another possibility could be that dicted to become reached not before several hours of power applithe contaminants are residues of 6E10, either the intact antibody cation and the positions of Aβ peptides at steady-state were at or fragments. The presence of sAPPα and 6E10 after IP of Aβ has about 4.7 and 5.2 mm from the anode, respectively. previously been experimentally observed by Haußmann et al. (refer to the Supporting Information of22). Thus, the carrier components for IEF were selected to accomplish an efficient removal of such contaminants. Simulations revealed that sAPPα would focus in the Glu-(Asp-His) boundary while 6E10 would position itself in the cSer-Arg transition (Figure 2B). It is well known that Aβ peptides are prone to aggregate. However, at pH values below 4 or above 8 the monomeric form of Aβ peptides dominates.39 The pH of the IEF buffer was adjusted with TFA as additive and the consequences of the TFA addition on the focusing dynamics were studied with simulations. Figure 3 shows

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Figure 3. IEF of 1 µM Aβ 1-38 and 1-40 and 0.1 µM sAPPα and 6E10 in a mixture composed of Glu, Asp-His, cSer, and Arg (5 mM each) and (A) 0.05% and (B) 0.1% TFA. The plots show the computer predicted distributions of ampholytes and TFA, peptides, and pH after 10 min application of 74.0 V. The initial pH and conductivities were (A) 4.19 and 0.058 S/m, and (B) 3.21 and 0.11 S/m. The anode is on the left. IEF and micropillar-MALDI-MS of Aβ peptides. As a first comparison of computer predicted and experimental data, standard components (Aβ 1-38 and Aβ 1-40) were focused by IEF in the microchannel and MALDI-MS analysis was performed directly from the open channel. Compared with the predictions from the simulations (Figure 3), it was noticed that the Aβ focus had shifted somewhat more towards the cathode. It was observed that the pH of the Aβ standard solutions were lower than theoretically predicted, probably due to the presence of acids and salts added during the preparation of the standards. The lower pH indicates an excess of acid, which accumulates at the anode, and can thus explain the position shift of the Aβ zone toward the cathode. When 0.05% or 0.1% TFA was included in the IEF buffer, the highest Aβ peptide concentration (relative to the internal standard) was found at positions 7 and 8 mm from the anode, respectively. In the other parts of the channel, no or an insignificant analyte signal was obtained (data not shown). In the approach where micropillar MALDI targets were utilized, the focused zone can be sampled directly from the channel and deposited onto a pillar as depicted in Figure 1. An advantage of using elevated microtargets is that the sample and matrix are confined on top of the pillar, and spreading to surrounding surfaces, which is sometimes observed with conventional MALDI targets, is avoided.19

Figure 4. Micropillar-MALDI-MS spectra of 5 µM Aβ 1-38 and 1-40 in 5 mM Glu-(Asp-His)-cSer-Arg with 0.05% TFA. 2.5 nL were sampled and deposited on 100 µm pillars. (A) Sampling without voltage application. (B) Sampling 7 mm from the anode after IEF (15 min, 74 V). To estimate the concentration efficiency of the IEF procedure, the S/N values obtained with micropillar-MALDI-MS analysis of Aβ 1-38 and Aβ 1-40 without (Figure 4A) and after IEF (Figure 4B) were compared. Performing the IEF procedure resulted in an increase of the S/N for the Aβ peptides by a factor of at least 10. It was observed that the samples contain a large portion of oxidized species (Figure 4), with oxidation likely occurring at methionine35 in the peptides. Since it was found that the use of TFA promoted oxidation, it is likely that the oxidation can be attributed to sample processing. This can, however, be accounted for by summation of the peak areas for the oxidized and non-oxidized form of each peptide. CSF and plasma samples. The developed method was used for analysis of Aβ peptides after IP in CSF and plasma. For these samples, a better agreement with the simulation results than for the standard peptides was obtained, probably because salts (believed to cause the position shift towards the cathode for the standards) were removed during IP, and pure water was used as IP elution solvent. For a system with 0.1% TFA in the IEF buffer, the location of the focused Aβ peptides was at a position of 7 mm from the anode, as predicted by simulations. The purification ability of the IEF method was investigated using a CSF sample for which a proper crystallization with the MALDI matrix was difficult to obtain when the IP eluate was deposited onto a micropillar. The IP eluate was vacuum centrifuged to dryness, redissolved in 1 µL of 5 mM Glu-(Asp-His)cSer-Arg with 0.1% TFA, and 2.5 nL of this solution were deposited on a micropillar. No Aβ peptides could be detected by MALDI-MS (Figure 5A), which is believed to be caused by contaminants present in the sample. However, when the microchannel was filled with 75 nL of the same sample eluate followed by IEF and sampling of the focused zone, micropillar-MALDI-MS analysis revealed the presence of several Aβ peptides (Figure 5B). This demonstrates the effectiveness of the IEF purification protocol.

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Figure 5. Micropillar-MALDI-MS spectra of an immunoprecipitated CSF sample in 5 mM Glu-(Asp-His)-cSer-Arg with 0.1% TFA. 2.5 nL were sampled and deposited on 100 µm pillars. (A) Sampling without voltage application. (B) Sampling 7 mm from the anode after IEF (10 min, 74 V). Another aim of including a purification step was to enable the use of a larger portion of the available sample without risking a loss of signal in MALDI-MS analysis due to contaminants present in the IP eluate. The IP work-up of 800 µL of body fluid (CSF or plasma) leads to a concentrate 1.5 µL in volume, yielding a theoretical concentration factor of around 500. Quantification studies utilizing immunoassays have reported values for Aβ 1-42 in CSF and plasma of AD patients in the pg/mL range (mean values in CSF: 36-709 pg/mL and in plasma: 31-140 pg/mL).8 Thus, according to this information, the maximum achievable Aβ 1-42 concentration after the IP enrichment used is in the ng/mL range. In Figure 6A, a MALDI spectrum after deposition of 5 nL of IP eluate (after IP of Aβ peptides from a CSF patient sample) onto a micropillar target is shown, representing the best obtained signal for this particular sample without the IEF purification step. It could be anticipated that using a larger portion of the available IP eluate would increase the signal in MALDI-MS. Using open microchannels, sample volumes larger than the channel volume can be analyzed by utilizing evaporation of the solvent during the application of sample. Loading the 1.5 µL IP eluate into the 75 nL open channel corresponds to an additional concentration increase by a factor of 20. Finally, together with IEF which provides a 10fold concentration, the overall sample enrichment could theoretically become 105-fold. Figure 6B shows a MALDI spectrum obtained with 1.5 µL of the same IP eluate. The eluate was applied to a microchannel, IEF buffer was added, and 5 nL were sampled after focusing. It is clear that the existing peptide signals were increased, and several other Aβ peptides were detected, including Aβ 1-42. Without the IEF step, it would not have been possible to detect these components. Thus, it is reasonable to assume that the complete scheme of IP-IEF-micropillar-MALDIMS is capable of detecting pg/mL concentrations of Aβ peptides. Quantification using labelled peptides was outside the scope of this paper, but would be an interesting topic for a future study. The method was applied also to blood plasma samples. In micropillar-MALDI-MS analysis of 5 nL of the IP eluate no Aβ peptides could be detected (Figure 7A). However, when 1 µL of the same IP eluate was applied to the open microchannel, IEF buffer added and 5 nL containing the focused Aβ peptides were sampled after IEF, several Aβ isoforms were detected (Figure 7B).

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Figure 6. Micropillar-MALDI-MS spectra of an immunoprecipitated CSF sample. 5 nL were sampled and deposited on 200 µm pillars. (A) Sampling of IP eluate (in water), no IEF. (B) 1.5 µL supplied to microchannel, IEF in 5 mM Glu-(Asp-His)-cSerArg with 0.1% TFA (10 min, 74 V) and sampling 7 mm from anode.

Figure 7. Micropillar-MALDI-MS spectra of an immunoprecipitated plasma sample. 5 nL were sampled and deposited on 200 µm pillars. (A) Sampling of IP eluate (in water), no IEF. (B) 1 µL supplied to channel, IEF in 5 mM Glu-(Asp-His)-cSer-Arg with 0.1% TFA (10 min, 74 V) and sampling 7 mm from anode. CONCLUSIONS We have shown that with a set of small and chemically welldefined carrier components and the aid of computer simulation a step-wise pH gradient for preconcentration and purification of Aβ peptides by IEF can be created. An accurate prediction of the location of the foci is facilitated by the use of simple ampholytes instead of complex mixtures of commercial carrier ampholytes. Herein, we chose to focus on the C-terminal species with a pI of 5.3, however, by selecting other carriers, the pH interval for focusing can easily be altered,23 leading to a number of potential applications of this approach. The presented semi-open instrumental setup enables concentrating a microliter sample volume into nanoliters: the evaporation from the open system can be exploited when sample is loaded into the channel, and hindered during focusing by applying a fluorocarbon liquid lid, which further provides access to the sample

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components. The purified Aβ peptides could be directly sampled from the open channel for subsequent analysis by micropillarMALDI-TOF-MS, which is shown to be a simple and straightforward way to combine these techniques. The IEF procedure enables concentration and purification of Aβ peptides after IP, and the complete scheme of IP-IEFmicropillar-MALDI-MS allowed the detection of Aβ peptides in CSF and plasma samples. The results presented in this methodological pilot study suggest that this technique could have the potential to contribute to AD related research aimed at developing an increased understanding of the disease mechanisms, exploring new treatment possibilities or enabling early AD diagnosis. Developing the instrumental setup to an automatable format and using the presented method on a larger set of samples is a potential topic for a future study.

ASSOCIATED CONTENT Supporting Information The Supporting Information contains two tables with input parameters used in computer simulations, and photographs taken after deposition of IP eluate onto micropillars, as described in the text. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Ute Haussmann, Hans Klafki and Jens Wiltfang (current address Universitätsmedizin Göttingen, Georg-August-Universität) are acknowledged for providing the magnetic beads used for IP and the IP protocol. Hans Klafki is further acknowledged for valuable discussions. Sanna-Kaisa Herukka (Institute of Clinical Medicine/Neurology, University of Eastern Finland) is acknowledged for the CSF samples. The EU project NaDiNe (contract no. 246513) is acknowledged for financial support. Part of the simulation work was supported by the Swiss NSF (grant no. 315230146161).

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