Analysis of Amino-Terminal Variants of Amyloid-β Peptides by

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Analysis of Amino-Terminal Variants of Amyloid‑β Peptides by Capillary Isoelectric Focusing Immunoassay Ute Haußmann,† Olaf Jahn,‡ Philipp Linning,§ Christin Janßen,† Thomas Liepold,‡ Erik Portelius,∥ Henrik Zetterberg,∥,⊥ Chris Bauer,⊗ Johannes Schuchhardt,⊗ Hans-Joachim Knölker,§,* Hans Klafki,*,† and Jens Wiltfang† †

LVR-Klinikum Essen, Department of Psychiatry and Psychotherapy, Faculty of Medicine, University of Duisburg-Essen, D-45147 Essen, Germany ‡ Max-Planck-Institute of Experimental Medicine, Proteomics Group, D-37075 Göttingen, Germany § Department of Chemistry, Technische Universität Dresden, D-01069 Dresden, Germany ∥ Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, 431 80 Mölndal, Sweden ⊥ UCL Institute of Neurology, Queen Square, London, U.K. ⊗ MicroDiscovery GmbH, D-10405 Berlin, Germany S Supporting Information *

ABSTRACT: Here we present a novel assay for the separation and detection of amino-terminal amyloid-β (Aβ) peptide variants by capillary isoelectric focusing (CIEF) immunoassay. Specific amino-terminally truncated Aβ peptides appear to be generated by β-secretase (BACE1)-independent mechanisms and have previously been observed in cerebrospinal fluid (CSF) after BACE1 inhibitor treatment in an animal model. CIEF immunoassay sensitivity is sufficient to detect total Aβ in CSF without preconcentration. To analyze low-abundance amino-terminally truncated Aβ peptides from cell culture supernatants, we developed a CIEF-compatible immunoprecipitation protocol, allowing for selective elution of Aβ peptides with very low background. CIEF immunoassay and immunoprecipitation mass spectrometry analysis identified peptides starting at residue Arg(5) as the main amino-terminal Aβ variants produced in the presence of tripartite BACE1 inhibitor in our cell culture model. The CIEF immunoassay allows for robust relative quantification of Aβ peptide patterns in biological samples. To assess the future possibility of absolute quantification, we have prepared the Aβ peptides Aβx‑10, Aβx‑16, and Aβ5‑38(D23S) by using solid phase peptide synthesis as internal standards for the CIEF immunoassay. myloid β (Aβ) peptides are produced from the amyloid precursor protein (APP) by consecutive proteolytic cleavages executed by β- and γ-secretases. They are the major proteinaceous constituents of the typical amyloid plaques found in the brains of Alzheimer’s disease (AD) patients,1,2 and specific oligomeric forms are believed to have neurotoxic properties.3−5 Pharmacological reduction of Aβ production is considered a potential causal AD therapeutic strategy, and small molecule β- and γ-secretase inhibitors have been tested in clinical trials (reviewed in ref 6). Several Aβ variants differing in their amino- and carboxytermini are present in biological fluids, such as cerebrospinal fluid (CSF) and blood.7,8 In AD patients, a selective reduction of those Aβ peptides ending at residue 42 (Aβ42) in CSF represents a well-established biomarker (recently reviewed in refs 9 and 10).

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© XXXX American Chemical Society

Antibody-based assays are commercially available for the quantification of carboxy-terminal Aβ variants ending at amino acid residues 42, 40, and 38. Mass spectrometry provides an alternative method for measuring Aβ peptides. In combination with preanalytical sample preparation by immunoprecipitation or solid phase extraction, it has been applied successfully to the characterization of Aβ patterns in human CSF.11,12 Amino-terminally truncated Aβ peptides represent more than 60% of the Aβ species in AD brains.13 “Ragged NH2-termini” of Aβ were discovered as early as 1985 in amyloid plaque cores isolated from AD brain tissue.2 Later studies revealed that in particular Aβs starting at Glu(3), which is modified to Received: April 15, 2013 Accepted: July 27, 2013

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pyroglutamate, are highly abundant in neuritic plaques of AD patients.14 These modified pGlu(3) Aβ peptides are more prone to aggregation than variants starting with Asp(1).15 Taken together, amino-terminally truncated Aβ variants may be of high pathophysiological relevance. Furthermore, specific alterations in the ratio of Aβ peptides starting at Asp(1) relative to those starting at Ala(2) in blood plasma have been proposed to represent a novel diagnostic biomarker candidate.16 Treatment of cell cultures and dogs with BACE1 inhibitors significantly reduced Aβs starting at Asp(1), while aminoterminally truncated variants such as Aβ5‑40 increased.17−19 The analysis of specific changes in the Aβ pattern was thus proposed to be of potential value for monitoring treatment effects in clinical trials.19 Since the amino acid residues Asp(1) and Glu(3) of the Aβ sequence have charged side chains, amino-terminal variants of the peptides can be separated by isoelectric focusing. We report here on the development of a novel capillary isoelectric focusing-(CIEF-) immunoassay for the detection and discrimination of amino-terminal Aβ variants in cell culture supernatants and other biological samples. The assay is a novel application of the automated NanoPro 1000 platform. This CIEF immunoassay technology was originally developed by O’Neill et al. for the analysis of phosphorylated and unphosphorylated forms of the extracellular signal regulated kinase (ERK1/2).20 Briefly, the sample is subjected to isoelectric focusing in a microcapillary. In a UV light-catalyzed reaction the focused proteins are covalently bound to the capillary inner surface, followed by an immunodetection protocol analogous to Western blot detection with a chemiluminescence readout.20 In the present study, the novel Aβ CIEF immunoassay was used for relative quantification of the biochemical effects of a membrane-tethered tripartite BACE1 inhibitor on the Aβ species released in cell culture experiments. Coupling of a pharmacophoric group (BACE1 inhibitor) to a membrane anchor via a spacer in a tripartite structure21 significantly improved the potency of prototype BACE1 inhibitors, presumably by achieving high local concentrations in lipid rafts.18,22,23 The CIEF immunoassay revealed substantial changes in the relative abundance of specific amino-terminal Aβ-variants upon treating APP-transfected SH-SY5Y cells with a tripartite BACE1 inhibitor. The observed Aβ peptides were further characterized and identified by 2DE Western blot analysis and immunoprecipitation followed by mass spectrometry.

Figure 1. Detection of synthetic Aβ peptides by CIEF immunoassay: (A) detection of synthetic Aβ1‑40, Aβ2‑40, Aβ4‑40, and Aβ5‑40 (25 ng/mL each). Approximately 0.4 μL of sample were loaded onto each capillary. Signals were detected with mAb 6E10 (2 μg/mL), after 30 s exposure. The chemiluminescence signals are plotted against the pI, which is calculated automatically for each capillary from fluorescent pI standards included in the samples. (B) Aβ2‑40 (100 ng/mL), Aβ3‑40 (200 ng/mL), and AβpGlu3‑42 (200 ng/mL) are not resolved in the CIEF immunoassay. Approximately 0.4 μL of sample was loaded onto each capillary. Signals were detected with mAb 6E10 (2 μg/mL), with an exposure of 30 s.

Table 1. Comparison of Observed and Theoretical pI Values for Amino-Terminal Aβ Variants

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RESULTS AND DISCUSSION Aβ Peptides with Different Amino-Termini Can Be Resolved and Detected by CIEF Immunoassay. Synthetic Aβ1‑40, Aβ2‑40, Aβ3‑40, AβpGlu3‑42, Aβ4‑40, and Aβ5‑40 were dissolved in bicine-CHAPS buffer and subjected to the NanoPro CIEF immunoassay. Each sample contains fluorescent pH standards, which are used to define a pH calibration curve for each capillary, allowing assignment of a pI value to each chemiluminescence signal.20 As shown in Figure 1, Aβ1‑40 was detected at pH 5.3 while variants starting with Ala(2), Glu(3), or pyroglutamate(3) (pGlu(3)) were detected at pH 6.0. Aβ peptides starting with Phe(4) or Arg(5) exhibited a pI of approximately 6.5. The observed pIs were in reasonable agreement with theoretical values (Table 1). The theoretical pI values calculated by ProteinCalculator v3.3 (www.scripps.edu/ ∼cdputnam/protcalc.html) differed by 0.28−0.5 pH units

peptide

observed pIa

Aβ1‑40 Aβ2‑40 Aβ3‑40 Aβ4‑40 Aβ5‑40

5.33 5.98 5.96 6.44 6.47

SDb (n) 0.013 0.010 0.005 0.006 0.005

(10) (11) (7) (5) (5)

theoretical pIc 5.59/5.31 6.25/5.78 6.25/5.78 6.77/6.27 6.77/6.27

The observed pI value is the mean pI value measured in at least five independent experiments. In each experiment, the peptide was loaded at least in two capillaries. bSD: standard deviation of the observed pI values in at least five independent experiments. The number n of experiments is given in parentheses. cFor comparison, theoretical pI values are listed that were calculated with two different tools: ProteinCalculator v 3.3 (www.scripps.edu/∼cdputnam/protcalc. html)/ProtParam (web.expasy.org/protparam/).24 a

from those calculated by ProtParam (web.expasy.org/ protparam/),24 which is presumably due to different algorithms employed or usage of different pK values for charged groups.25 Whether or not the differences in signal intensities observed with different amino-terminal Aβ variants (Figure 1A) were related to specific properties of the mAb 6E10 or to the synthetic peptide preparations used is not clear at this stage. In contrast to amino-terminal variants of Aβ peptides, carboxyB

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Figure 2. “BACE1 inhibitor-resistant” Aβ variants display shifted pI values. (A) SH-SY5Y cells overexpressing APPwt were incubated for 72 h with either 100 nM tripartite BACE1 inhibitor or 0.1% DMSO (vehicle control). Aβ peptides were immunoprecipitated from cell culture supernatants with mAb 6E10 and analyzed by CIEF immunoassay or 2DE Western blot. In both cases, mAb 6E10 was used for detection. (B) Relative quantification of peak areas of the three Aβ pI variants in controls and treated cell culture supernatants. The peak area (AUC) of each of the three Aβ pI variants within a single IEF capillary was calculated as % of the sum of the three of them (relative peak area). The means and standard deviations from n = 6 treated culture dishes and n = 4 DMSO controls from four independent experiments are shown. Statistical analysis (two-tailed t test) was carried out with data from 120 s exposures. Under these conditions, none of the signals was saturated. To better visualize the very small relative peak areas at pH 6.0 and 6.5 in the control samples, a logarithmic scale was chosen for the y-axis. ***p-value 99.9% 98.6% >99.9% >99.9% >99.9% >99.0% >99.0%

4.20 5.38 5.22 6.14 6.75 6.71 6.47 7.08

SDb (n)

theoretical pIc

0.044 0.004 0.005 0.010 0.009 0.007 0.002 0.006

4.70/4.54 5.69/5.32 5.69/5.32 6.25/5.76 6.77/6.28 6.77/6.28 6.77/6.27 7.56/7.03

(7) (4) (4) (4) (3) (3) (3) (3)

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EXPERIMENTAL SECTION

Aβ Peptides. Synthetic Aβ peptides were either purchased from AnaSpec or synthesized by solid phase peptide synthesis. Detailed procedures for synthesis of peptides Aβ1‑10, Aβ2‑10, Aβ3‑10, Aβ1‑16, Aβ2‑16, Aβ3‑16, Aβ5‑38, and Aβ5‑38(D23S) are given in the Supporting Information. Recombinant Aβ1‑40 was purchased from rPeptide. Cell Culture. SH-SY5Y cells stably overexpressing APP695 with wild-type sequence carrying an amino-terminal Myc tag and a carboxy-terminal Flag tag29 were kindly provided by L. Münter and G. Multhaup (current address, McGill University Montreal, Canada). The cells were cultured in D-MEM/F12, supplemented with 10% fetal calf serum, 2 mM L-glutamine, Non-Essential Amino Acids (Gibco), and 50 μg/mL Hygromycin B. Cultures were grown at 37 °C, 5% CO2. Inhibitor Treatments. The tripartite BACE1 inhibitor used in this study corresponds to compound 6a as described by Linning et al.23 The tripartite inhibitor was initially dissolved in DMSO at a concentration of 10 mM and stored in aliquots at −20 °C. For the cell culture experiments, compound dilutions were prepared in 100% DMSO that were added freshly to cell

a

The observed pI value is the mean pI value measured in at least three independent experiments. In each experiment, the peptide was loaded at least in two capillaries. bSD: standard deviation of the observed pI values in at least three independent experiments. The number n of experiments is given in parentheses. cFor comparison, theoretical pI values are listed that were calculated with two different tools: ProteinCalculator v 3.3 (www.scripps.edu/∼cdputnam/protcalc. html)/ProtParam (web.expasy.org/protparam/)24

Synthesis protocols, electropherograms, and peptide sequences are shown in the Supporting Information (Figure S7). All tested peptides were successfully detected in the CIEF immunoassay with mAb 6E10. The signal strength increased with increasing peptide length: signals of the longest peptide variant (Aβ5‑38(D23S)) were comparable to other synthetic full length Aβ peptides, while Aβx‑16 and Aβx‑10 variants required F

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culture medium. The final concentration of DMSO in the experiments and controls was 0.1%. The cell culture medium was replaced with fresh medium containing the compound or 0.1% DMSO. For the BACE1 inhibitor treatment, 5 × 106 SHSY5Y cells were seeded 24 h before treatment in a 10 cm cell culture dish or a 96 well plate with 2 × 105 cells/cm2. The compounds were diluted in medium and added to the cells as described above. Supernatants were collected after 72 h, centrifuged for 5 min at 500g, 4 °C, and stored at −20 °C. Immunoprecipitation for Western Blot and CIEF Immunoassay. Immunoprecipitation of Aβ peptides from cell culture supernatants was performed as described in ref 18 with mAb 6E10 (Covance) as capture antibody. For samples to be analyzed by capillary isoelectric focusing, an additional washing step with 20 mM bicine, pH 7.6, 0.6% CHAPS, 3 min at room temperature, was included, followed by elution in 25 μL of 20 mM bicine, pH 7.6, 0.6% CHAPS for 5 min at 95 °C. A detailed description of the experimental procedures is given in the Supporting Information. SDS-PAGE and Western Blot. One-dimensional and twodimensional electrophoresis and Western blots were performed essentially as described in refs 8 and 18. The experimental procedures are described in detail in the Supporting Information. The antibodies mAb 6E10 (Covance) or mAb 1E8 (Nanotools), both recognizing the amino-terminus of Aβ, were used for blot detection. CIEF Immunoassay (NanoPro 1000 Technology). Automated capillary isoelectric focusing immunoassays were performed with a NanoPro 1000 system (ProteinSimple). This CIEF immunoassay technology was developed by O’Neill et al.20 IP eluates or synthetic Aβ peptides diluted in 20 mM bicine, pH 7.6, 0.6% CHAPS were mixed 1:4 with G2 Premix containing a pH gradient ranging from pH 5−8 (nested, with a pH 2−4 plug), fluorescent pH standards (Standard Ladder 3 (pH 4.9, 6.0, 6.4, 7.0, 7.3) + pH standard 5.5) and a DMSO inhibitor mix (all reagents were obtained from ProteinSimple). Samples, primary antibody (mAB 6E10, Covance, 1:500 (2 μg/ mL) in ProteinSimple antibody diluent), secondary antibody (biotinylated antimouse, ProteinSimple, 1:100), streptavidincoupled horseradish peroxidase (ProteinSimple, 1:100), and a luminol/peroxide mix were loaded onto a 384 well sample plate, and the automated assay was programmed in the Compass software (ProteinSimple). The samples were loaded into the capillaries for 25 s. Each capillary measures 5 cm in length with an inner diameter of 100 μm. Analytes were separated for 40 min with 21 000 μW. Analytes and standards were immobilized within 100 s, followed by two washing steps (load, 20 s; soak, 150 s). The primary antibody was loaded within 2 s and incubated for 120 min. After two washing steps, the secondary antibody was loaded for 2 s and incubated for 60 min. After two washing steps, the streptavidin-HRP conjugate was loaded and incubated as described for the secondary antibody. After two final washing steps, luminol/peroxide was loaded for 2 s, and chemiluminescence signals were detected at six exposure times (30, 60, 120, 240, 480, and 960 s). CIEF Immunoassay Data Analysis. The pI values of the analytes were calculated by the Compass software using the positions of the fluorescent pH standards in each capillary. Chemiluminescence signals were automatically plotted against the pH gradient. Analyte peaks were named, and the relative peak area of each peak was automatically calculated. To determine a suitable exposure time for data analysis, the linear

range of all analyte signals was determined by manually plotting peak areas against exposure time. Screening of Aβ Variants by ImmunoprecipitationMass Spectrometry. The immunoprecipitation and matrixassisted-laser-desorption/ionization time-of-flight/time-of-flight (MALDI TOF/TOF) analysis was performed as described elsewhere.19 Briefly, 4 μg of the anti-Aβ antibodies 6E10 and 4G8 (Signet Laboratories, Dedham, MA) was separately added to 50 μL each of magnetic Dynabeads M-280 Sheep AntiMouse IgG (Invitrogen, Carlsbad, CA). The mAB 6E10 and 4G8 antibody-coated beads were mixed and added to the cell media to which 0.025% Tween20 in phosphate-buffered saline (pH 7.4) had been added. Mass spectrometry measurements were performed using a Bruker Daltonics UltraFleXtreme MALDI TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). For each peak the sum of the heights for the three highest isotopes were averaged followed by normalization against the sum for all the Aβ peaks in the spectrum. It should be noted that a relative quantification cannot be interpreted as a direct reflection of an absolute or relative abundance of an isoform since the ionization efficiency might be different for different isoforms and since different isoforms are more hydrophobic than others. Mass Spectrometric Detection of Lys-C Cleavage Products. The washing protocol after immunocapture (see above and ref 18) was adjusted for mass spectrometry analysis: After two washing steps with PBS, two washing steps with 50 mM NH4HCO3 and one step with ultrapure H2O (all 5 min at room temperature), immunoprecipitated Aβ peptides were eluted for 5 min at 95 °C with 50 μL of 0.05% n-octyl β-Dglucopyranoside (OGP), a nonionic detergent with performance-enhancing effects on both enzymatic digestion and MALDI-MS.30 To determine the mass of the intact Aβ peptides prior to digestion, the volume was reduced to ∼10 μL in a vacuum centrifuge, and 0.3 μL were spotted onto an AnchorChip target (Bruker Daltonics, Bremen, Germany) precoated with α-cyano-4-hydroxycinnamic acid (CHCA), let dry, and washed twice with ammonium dihydrogen phosphate (10 mM in 0.1% TFA). For enzymatic cleavage, 200 ng of endoproteinase Lys-C (sequencing grade, Roche Diagnostics, Mannheim, Germany) were added to the remainder of the solution, and digestion was allowed to proceed overnight at 37 °C. Digestion was stopped by acidification with 1% TFA, and the Lys-C cleavage products were analyzed mass spectrometrically according to a standard CHCA-based thin-layer affinity method.30 Mass spectrometry was performed using an Ultraflex MALDI TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) as described earlier.30,31

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Notes

J. Schuchhardt and C. Bauer are full time employees of MicroDiscovery, Berlin, Germany. The authors declare no competing financial interest. G

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ACKNOWLEDGMENTS This work was funded by the grant PURE (Protein Research Unit Ruhr within Europe) from the State Government North Rhine-Westphalia, by the EU Grant NADINE (Contract No. 246513), by the German Federal Ministry for Education and Research (as a part of the BIOMARKAPD project in the JPND programme and by BioPharma-Neuroallianz Grant 161A120B/ 031A120B), and by the Swedish Research Council. The authors thank L. Münter and G. Multhaup (current address, McGill University, Montreal, Canada) for providing stably transfected SH-SY5Y cells, P. Lewczuk (University of Erlangen-Nuremberg, Germany) for providing CSF samples, B. Müller (LVRKlinikum Essen, University of Duisburg-Essen, Germany) for helpful discussions, and H. Kamrowski-Kruck for excellent technical assistance. We are grateful to A. Shevchenko (MPICBG, Dresden, Germany) for the measurement of the highresolution ESI mass spectra and to M. Gruner (TU Dresden, Germany) for the measurement of the NMR spectra.

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