Multi Epitope-Targeting Immunoprecipitation Using F(ab′) Fragments

Feb 8, 2013 - ... and Specificity for the Enhanced Detection of a Peptide with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectro...
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Multi Epitope-Targeting Immunoprecipitation Using F(ab′) Fragments with High Affinity and Specificity for the Enhanced Detection of a Peptide with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry Naoki Kaneko,† Takayuki Yoshimori,† Rie Yamamoto,† Daniel J. Capon,§ Takashi Shimada,‡ Taka-Aki Sato,†,‡ and Koichi Tanaka*,† †

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan ‡ Life Science Research Center, Shimadzu Corporation, Kanda-Nishikicho 1, Chiyoda-ku, Tokyo 101-8448, Japan § Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, California 94118, United States S Supporting Information *

ABSTRACT: Human plasma has been frequently studied using mass spectrometry for new biomarker discovery, although detection of low-abundance biological molecules can be challenging due to sample complexity and dynamic protein concentration ranges of plasma proteins. While immunoprecipitation coupled with mass spectrometric analysis is an essential method for overcoming this difficulty, its sensitivity can be insufficient to detect clinically relevant circulating biomarkers because of limited antibody affinity or specificity. To increase antibody affinity, we developed a strategy using a F(ab′) fragment coupled to polyethylene glycol. We produced hetero-F(ab′)-(PEG)24 beads composed of two monoclonal antiamyloid β antibodies (6E10 and 4G8) that are specific for different epitopes of amyloid β and assessed the detection limit of amyloid β1−28-spiked human plasma. In human plasma, the detection limit of amyloid β1−28 was 6.14 pM, which was 25- to 50-fold more sensitive than single IgG-protein G beads. In addition, an introduction of polyethylene glycol as a linker reduced nonspecific binding, leading to highly specific MS detection. Finally, the present IP method enabled the detection of endogenous amyloid β1−40 in 250 μL of human plasma with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technique provides a powerful approach for enhancing the sensitivity and specificity of immunoprecipitation (IP)-MS for detection of low-abundance peptides in plasma and has the potential to accelerate MS-based clinical applications.

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(CCK-8) over a concentration range of 0.05 to 2.5 ng/mL in 500 μL of hamster plasma following IP with protein G agarose binding to IgG and liquid chromatography−tandem mass spectrometry (LC−MS/MS).2 The purification using primary antibody binding to magnetic beads via an immobilized secondary anti-IgG antibody enabled the detection of glucose-dependent insulinotropic polypeptide isoforms at concentrations ranging from 3 to 350 pM in 1.9 mL of human plasma and brain natriuretic peptide (BNP)-32 with a detection limit of 150 pM in 1 mL of human plasma sample.3−5 Mass spectrometric immunoassay (MSIA) with affinity pipet tips coupled to antibody was reported as an approach to discover N- and C-terminal variants of parathyroid hormone and detected degraded forms of BNP-32 with a sensitivity of

mmunoprecipitation (IP) is a useful tool for the quick and simple purification of a target molecule from biological samples such as cell lysates, serum, plasma, and cerebrospinal fluid (CSF). In particular, IP is an essential purification step prior to mass spectrometry (MS) analysis of low abundance peptides in biological fluids, because interference from nontarget molecules should be avoided in MS analysis for the following reasons: (i) owing to a limited spectrum dynamic range of typically 102−104, the signal for low-abundance target peptides can be drowned out by strong signals from highabundance nontarget peptides; and (ii) a peak appearing in the vicinity of a target peptide presents difficulties for quantifying the exact mass of the target molecule and the use of tandem mass spectrometry (MS/MS) analysis.1 Recently, IP in combination with MS (IP-MS) has attracted significant attention for structure analysis and peptide quantification in biological fluid samples. Young et al. reported the first detection and quantitative analysis of the cholecystokinin octapeptide © XXXX American Chemical Society

Received: November 19, 2012 Accepted: February 8, 2013

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3.1 pM in 500 μL of human plasma.6,7 Thus, IP-MS has been implemented successfully for the detection of low-abundance peptides in plasma. Although antibodies are generally produced by immunization of animals such as rabbit and mouse, affinity maturation in vivo frequently fails to produce antibodies with sufficiently high sensitivity and specificity to allow purification of proteins present at low levels. However, different approaches have recently been reported that improve antibody affinity. Some researchers reported a method that selects high-affinity scFvs derived from phage or yeast antibody libraries followed by conversion into human IgGs.8,9 Lippow et al. employed a computational design and a single mutation to improve affinity and achieve a Kd of 30−52 pM.10 We previously demonstrated that incorporation of polyethylene glycol (PEG) into the antibody hinge region remarkably enhanced the antibody− antigen interaction affinity (Kd = 0.53−53 pM).11 The presence of PEG is thought to enhance the flexibility and extension of the hinge region to promote cooperative binding. We believe that this approach provides the antibody affinity necessary for detecting clinical biomarkers using IP-MS. Amyloid β (Aβ), a peptide involved in Alzheimer’s disease (AD) pathogenesis, is generated by cleavage of the amyloid precursor protein (APP), and the resulting Aβ1−42 fragment forms aggregates.12,13 Accumulation of aggregated Aβ peptides in brain tissue causes the formation of senile plaques,14 and the presence of Aβ1−40 and Aβ1−42 in cerebrospinal fluid (CSF) and blood was reported to be a potential AD biomarker by enzymelinked immunosorbent assay (ELISA). Blennow et al. demonstrated the diagnostic performance of CSF Aβ1−42 with 86% sensitivity and 89% specificity in discriminating between AD and normal aging.15 Luis et al. showed that a low ratio of serum Aβ1−42/Aβ1−40 is associated with mild cognitive impairment (MCI) and AD.16 On the other hand, MS has an advantage over conventional ELISA for the simultaneous detection of multiple peptides. Indeed, many N- and Cterminally truncated Aβ peptides present in cultured cell media and CSF could be detected by IP-MS with antiamyloid β (Aβ) antibodies.17−20 In MS analysis of human plasma, however, Aβ isoforms have not been detected to date, because Aβ1−42 and Aβ1−40 plasma concentrations were approximately 5-fold lower than those in human CSF.21,22 In addition, the total protein concentration in human plasma (62−83 mg/mL) is from 89- to 277-fold higher than that in human CSF (0.3−0.7 mg/mL).23,24 As such, it is apparent that the larger number of total proteins in plasma and the lower concentration of Aβ peptides make the detection of plasma Aβ peptides more challenging. Therefore, there is a need to enhance immunoaffinity and reduce nonspecific binding in IP for MS detection of plasma Aβ peptide. In the present study, we developed immunoaffinity beads specific for two different Aβ epitopes by coupling F(ab′) fragments via a PEG linkage. A F(ab′) fragment linked to PEG can bind a target antigen with greater flexibility and less steric hindrance for cooperative binding. We used these beads together with matrix-assisted laser desorption ionization-timeof-flight mass spectrometry (MALDI-TOF MS) to evaluate the limit of detection (LOD) of plasma spiked with Aβ peptides and demonstrate the improved purification and enhanced detection of Aβ peptide in human plasma.

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EXPERIMENTAL SECTION Fragmentation of 6E10 IgG1 and 4G8 IgG2b to F(ab′)2. Mouse monoclonal anti-Aβ antibodies (6E10 IgG1 and 4G8 IgG2b) were purchased from Covance (Princeton, NJ). The buffers containing the antibody were exchanged using Zeba Desalt Spin Columns (Pierce, Rockford, IL) with 10 mM citrate buffer, pH 6.0, containing 5 mM EDTA-2Na (ethylenediaminetetraacetic acid, disodium salt) and 4 mM Lcysteine, and 50 mM Tris-HCl, pH 8.8, for 6E10 and 4G8, respectively. 6E10 IgG1 (250 μg) was incubated with 750 μL of immobilized ficin (Pierce) at 37 °C for 24 h. 4G8 IgG2b (100 μg) was incubated with 500 ng of Lys-C (Wako Pure Chemical Industries, Osaka, Japan) at 37 °C for 2.5 h. The proteolytic fragments were concentrated in Amicon Ultra filtration devices (Millipore, Cork, IR), and 50 μL of each concentrate was subsequently separated by size exclusion chromatography (SEC), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. SEC was carried out using a Prominence HPLC system (Shimadzu Corp, Kyoto, Japan). Mobile phase, column temperature, and detection wavelength used were 50 mM phosphate buffer, pH 6.5, containing 300 mM NaCl and 5 mM EDTA, 25 °C, and 214/ 280 nm, respectively. Flow rates for 6E10 and 4G8 were 0.1 and 0.2 mL/min, respectively. The proteolytic fragments were applied to a TSKgel G3000SWXL [7.8 mm i.d. × 30 cm length] column (TOSOH Bioscience, Tokyo, Japan). Portions of the 6E10 and 4G8 fractions were then analyzed using 7.5−15% and 10−20% SDS-PAGE, respectively, under reducing or nonreducing conditions. Proteins were visualized using a Silver Staining kit (Invitrogen, Carlsbad, CA). Fractions corresponding to the F(ab′)2 fragment were concentrated in Amicon Ultra filtration devices (Millipore). Construction of F(ab′)-(PEG)n Beads. F(ab′)2 fragments were reduced by 30 and 10 mM cysteamine for 6E10 and 4G8, respectively, at 37 °C for 30 min in 50 mM phosphate buffer, pH 6.5, containing 5 mM EDTA. To remove cysteamine from the solutions, 6E10 and 4G8 F(ab′) fragments were dialyzed in 50 mM phosphate buffer, pH 6.5, containing 5 mM EDTA using a Slide-A-Lyzer MINI Dialysis Unit (Pierce). Dynabeads M-270 Amine (5 μL suspension containing 30 mg beads/mL, Invitrogen) were washed three times with 20 μL of phosphate buffered saline (PBS) and added to a mixture of 10 μL of PBS and 0.18 μL of 250 mM SM(PEG)n Cross-linkers (Pierce) in dimethyl sulfoxide or 10 μL of 4.5 mM sulfo-SMCC crosslinkers (Pierce) dissolved in PBS. After incubating at room temperature for 30 min in the dark, the beads were washed three times with 20 μL of PBS to remove unbound SM(PEG)n or sulfo-SMCC and then resuspended in 5 μL of PBS. The dialyzed F(ab′) fragments (0.5 μg) were added to the bead solution, incubated at room temperature for 30 min in the dark, and washed three times with 20 μL of PBS to remove unbound F(ab′) fragments. After unreacted maleimide groups on the beads were blocked with 4 mM L-cysteine in PBS, F(ab′)(PEG)n beads or F(ab′)-SMCC beads were suspended in 20 μL of PBS. The binding efficiency of F(ab′) fragments was determined by measuring the difference in the protein concentration of the F(ab′) fragment solution before and after coupling reactions using the Coomassie Plus Protein Assay (Pierce). The binding efficiencies for F(ab′)-(PEG)n and F(ab′)-SMCC beads were 75.1 ± 0.434% and 72.9 ± 5.43%, respectively. The respective amounts of F(ab′) immobilized to 150 μg of (PEG)n beads and SMCC beads were 376 ± 2.17 ng B

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and 364 ± 27.1 ng, which corresponds to the approximate number of binding sites, 7.51 ± 0.0434 pmol and 7.29 ± 0.543 pmol, respectively. Preparation of IgG-Protein G Beads. Dynabeads Protein G (5 μL of suspension containing 30 mg beads/mL, Invitrogen) were washed with the binding buffer and incubated with 0.75 μg of 6E10 IgG1 and/or 4G8 IgG2b in the binding buffer at room temperature for 10 min. After washing three times with the binding buffer, IgG-protein G beads were suspended in 20 μL of the binding buffer. For binding between IgG and protein G, phosphate buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.4) containing 1% n-octyl-β-Dthioglucopyranoside (OTG) was used as the binding buffer for cross-linked IgG-protein G beads and tris-buffered saline (TBS) containing 1% OTG for noncross-linked IgG-protein G beads. The cross-linking reaction of IgG to protein G was performed by incubating IgG-protein G beads (150 μg) with 25 μL of 5 mM bis[sulfosuccinimidyl] suberate (BS3 ) (Pierce) in phosphate buffer at room temperature for 30 min. After the cross-linking reaction was quenched by addition of 1.25 μL of 1 M Tris-HCl (pH 7.5) and incubation at room temperature for 15 min, the cross-linked IgG-protein G beads were washed with and resuspended in 20 μL of TBS-1% OTG. The binding efficiency between IgG and protein G in phosphate buffer-1% OTG and TBS-1% OTG were determined to be 76.9 ± 2.87% and 75.2 ± 3.30%, respectively, using the same method as that for the F(ab′) fragments. The amount of IgG binding to 150 μg of protein G beads in cross-linked and noncross-linked IgG protein G beads was 577 ± 21.5 ng and 564 ± 24.8 ng, corresponding to the number of binding sites, which was approximately 7.69 ± 0.287 pmol and 7.52 ± 0.330 pmol, respectively. The numbers of binding sites on all types of tested immunoaffinity beads (F(ab′)-(PEG)n, F(ab′)-SMCC, and cross-linked and noncross-linked IgG-protein G) were shown to be almost equal. Immunoprecipitation of Aβ Peptides. Aβ1−28 (AnaSpec, San Jose, CA), Aβ1−42 (Sigma, St. Louis, MO), and human plasma (C.C. Biotech Corp., San Diego, CA) were diluted with TBS-1% OTG. The human plasma has been tested for HIV 1/ 2, HIV−Ag or HIV(PCR), HBsAg, HCV, HCV(PCR), and RPR by an FDA approved method and found to be nonreactive. The final concentration of plasma was 5% in TBS-1% OTG with or without spiked Aβ1−28. The plasma sample was subsequently pretreated with Protein G Plus Agarose (Pierce) at 4 °C for 30 min. After 150 μg of beads for each immunoaffinity medium, F(ab′)-(PEG)n, F(ab′)-SMCC, or cross-linked and noncross-linked IgG-protein G beads were washed three times with TBS-1% OTG, they were incubated with Aβ1−28 or Aβ1−42 in 10 μL of TBS-1% OTG or the plasma sample at 4 °C for 1 h. For the ELISA evaluation, the volume of TBS-1% OTG was 10, 100, or 1000 μL. The immunoaffinity media were then washed three times with TBS-1% OTG. For purification from the plasma sample, the washing was performed two additional times. After washing with H2O, Aβ peptides were eluted twice with 5 μL of 3 mM HCl for the detection of Aβ1−28 and Aβ1−42 or once with 5 μL of 15 mM HCl for the detection of endogenous plasma Aβ peptide. The eluted sample (1 μL) was immediately applied onto the MALDI plate. Aβ ELISA. The Aβ1−40 concentration in human plasma was measured using the human β amyloid (1−40) ELISA Kit Wako II (Wako) according to the manufacturer’s protocol. To determine the amount of Aβ1−42 in the eluate from affinity

beads, the following steps were performed. In total, 96 well microtiter plates (Maxisorp, Nalge Nunc, Rochester, NY) were coated with 100 μL of mouse monoclonal anti-Aβ antibody 4G8 in carbonate buffer (16 mM Na2CO3, 34 mM NaHCO3, pH 9.6). After incubation at 4 °C overnight, the plates were blocked with 20% Blocking One (Nacalai Tesque, Kyoto) in TBS containing 0.1% Tween-20 (TBST) at room temperature for 2 h. Eluted immunoprecipitation samples were diluted 100fold with TBST containing 5% Blocking One, and the Aβ1−42 standard range of 98 pg/mL to 25 ng/mL was diluted serially with TBST. Diluted samples and Aβ1−42 standard were added to the antibody-coated plates and incubated at room temperature for 1 h. After washing with TBST, 100 μL of mouse monoclonal anti-Aβ antibody 6E10 conjugated with HRP (Covance), diluted 1:2 000 in TBST containing 5% Blocking One were added to the plates and incubated at room temperature for 1 h. After the plates were washed with TBST, 3,3′,5,5′-tetramethylbenzidine substrate solution (Nacalai Tesque) was added to the plates and allowed to develop at room temperature for 15 min. After the reaction was stopped with 1 N H2SO4, the absorbance at 450 nm with a 595 nm reference was measured using an Infinite M200 PRO Multiwell reader (TECAN, Männedorf, Switzerland). MALDI-TOF MS Analysis. MALDI mass spectra were obtained using an AXIMA performance MALDI-TOF mass spectrometer (Shimadzu/KRATOS, Manchester, U.K.) equipped with a 337 nm nitrogen laser in the positive ion linear mode. MS spectra were generated from the accumulation of 2 500 laser shots from 100 different spots. MS/MS analysis for identification of plasma Aβ1−40 was obtained using an AXIMA resonance MALDI-TOF mass spectrometer (Shimadzu/ KRATOS) in the positive ion mode, and MS/MS fragments were generated by collision induced dissociation (CID) with helium and argon gas. For the MALDI matrixes, α-cyano-4hydroxycinnamic acid (CHCA), purchased from LaserBio Laboratories (Sophia-Antipolis Cedex, France), was used for AXIMA performance, and 2,5-dihydroxybenzoic acid (DHB), purchased from LaserBio Laboratories (Sophia-Antipolis Cedex), for AXIMA resonance. The matrix solutions were prepared by dissolving 0.5 or 5 mg of the matrix compounds in 1 mL of 50% v/v acetonitrile containing 0.1% v/v trifluoroacetic acid. For MALDI-TOF MS analysis, 1 μL of the sample solution or 15 mM HCl as a negative control was mixed with an equivalent amount of the matrix solution on the target plate and then dried at room temperature. For measurement of human plasma with or without spiked Aβ1−28, 1 μL of 2% methanediphosphonic acid (MDPNA) in water was used as a matrix additive. The signal-to-noise ratio (S/N) was determined using Launchpad version 2.9.1 software (Shimadzu, Kyoto, Japan). The LOD was established with a S/N of 3:1. All reported m/z represent the average peak of the protonated signal [M + H]+. The m/z values were calibrated with 1 pmol of human angiotensin II (m/z 1047.2), 1 pmol of human ACTH fragment 18−39 (m/z 2466.7), and 5 pmol each of bovine insulin oxidized β-chain (m/z 3497.0) and bovine insulin (m/z 5734.6) as an external standard. Peak lists were created from the generated MS/MS spectra by Launchpad version 2.9.1 software, and peaks corresponding to background peaks present with the negative control were removed, followed by analysis using Mascot software version 2.4 (Matrix Science). The Mascot search parameters were as follows: no enzyme, SwissProt database with species limitation (only human), and C

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binding by multiple 6E10 F(ab′)s. The aggregated form was confirmed using SDS-PAGE followed by silver staining (Figure S-3 in the Supporting Information). A fixed amount (100 ng) of Aβ1−42 present in 10, 100, and 1000 μL of TBS-1% OTG was captured by each bead type and then eluted. OTG was used as a detergent owing to its effectiveness in suppressing nonspecific binding and low ionization in MALDI-TOF MS.26 The eluates were subjected to sandwich ELISA to quantify the recovered Aβ1−42 (Figure 2A). The amounts of Aβ1−42 recovered using

tolerances of 0.1 and 0.4 Da for the precursor and fragment ions, respectively.

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RESULTS AND DISCUSSION Construction of iso-F(ab′)-(PEG)n Beads. The strategy for conjugating F(ab′) fragments to magnetic beads via PEG is shown in Figure 1. SM(PEG)n, which is a heterobifunctional

Figure 1. Development of 6E10 F(ab′)-(PEG)n beads: schematic illustration of F(ab′)-(PEG)n bead synthesis. The amino group on the magnetic beads was first reacted with the NHS ester of SM(PEG)n, and then the maleimide group of (PEG)n was linked to the thiol group in the F(ab′) hinge region.

cross-linker with an N-hydroxysuccinimide (NHS) ester and a maleimide group, was used to cross-link the amino group of magnetic beads with the thiol group in the hinge region of the F(ab′) fragments. Mouse antihuman Aβ IgG1 (6E10), which recognizes an epitope within amino acids 3−8 of the Aβ peptide, was used to generate F(ab′) fragments. 6E10 was digested by immobilized ficin and separated using size exclusion chromatography (SEC) (Figure S-1A in the Supporting Information). The digested antibody in each column fraction was subsequently confirmed by SDS-PAGE under reducing and nonreducing conditions (Figure S-1B in the Supporting Information). The 6E10 F(ab′)2 fragment eluted in fraction number (Frac.) 24−29 with bands corresponding to typical molecular weights of nonreduced and reduced F(ab′) 2 fragments (approximately 100 and 25 kDa, respectively). Cysteamine was used to reduce the disulfide bonds between cysteine residues in the F(ab′)2 fragment hinge region, which was confirmed by SEC (Figure S-2 in the Supporting Information). Magnetic beads were conjugated to NHS of SM(PEG)n, followed by conjugation of the 6E10 F(ab′) fragment to the SM(PEG)n maleimide group. Two different PEG units (n = 2 or 24) were used to evaluate their affinity for Aβ peptides. Comparison of Aβ1−42 Levels Captured by iso-F(ab′)(PEG)n Beads and IgG-Protein G Beads. We evaluated the capture efficiency of 6E10 F(ab′)-(PEG)n beads by comparison with the value obtained for 6E10 IgG binding to protein Gcoupled magnetic beads. Aβ1−42 known to self-aggregate into oligomers25 was used as a target peptide, because aggregated Aβ1−42 has the multiepitope 6E10 that allows cooperative

Figure 2. Comparison of Aβ1−42 levels captured by 6E10 F(ab′)(PEG)n beads and 6E10 IgG-Protein G beads. (A) Quantitative analysis of Aβ1−42 recovered by three types of beads. Aβ1−42 (100 ng) in 10, 100, or 1000 μL of TBS-1% OTG was recovered using 6E10 F(ab′)-(PEG)n beads (n = 2 and 24) and IgG-protein G beads and followed by sandwich ELISA. (B) LODs of Aβ1−42 recovered by each type of bead or pure Aβ1−42 applied directly onto the MALDI plate. Serial dilutions of Aβ1−42 in 10 μL of TBS-1%OTG were recovered using 150 μg of each different type of bead, and 1 μL from 10 μL eluate was applied to MALDI-TOF MS. A volume of 1 μL from 10 μL of serially diluted pure Aβ1−42 dissolved in 3 mM HCl was directly applied onto the MALDI plate for determination of a reference LOD. The LOD was defined as the amount that yields a measurable peak with S/N ≥ 3.

6E10 F(ab′)-(PEG)n (n = 2 and 24) beads were from 5.9- to 8.5-fold and 5.6- to 6.9-fold higher, respectively, than 6E10 IgGprotein G beads. Although in general there was a decreased amount of Aβ1−42 binding to beads in larger volumes, the 6E10 F(ab′)-(PEG)24 beads had the smallest decrease in binding among the three types of beads tested, which indicated it had the highest Aβ1−42 binding capacity in a volume of 1000 μL. To determine whether the higher binding ability of 6E10 F(ab′)(PEG)n beads leads to enhanced sensitivity in MS, we examined the LOD of Aβ1−42 (m/z 4515) by MALDI-TOF MS. While 6E10 IgG-protein G beads had a LOD of 100 pg, both 6E10 F(ab′)-(PEG)24 and 6E10 F(ab′)-(PEG)2 beads had a LOD of 25 pg, which was equal to that of pure Aβ1−42 applied directly to the MALDI plate (Figure 2B and Figure S-4 in the D

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G beads (LOD, 25 pg), whereas 4G8 F(ab′)-(PEG)24 beads had a LOD of 25 pg, which was equal to 4G8 IgG-protein G beads. Among the three types of F(ab′)-(PEG)24 beads, 6E10/ 4G8 F(ab′)-(PEG)24 beads (1:1, w/w) showed the lowest LOD of 2.5 pg, which was comparable to pure Aβ1−28 applied directly onto MALDI plates. The expected LOD for Aβ1−28 using 6E10/4G8 F(ab′)-(PEG)24 beads was approximately 15 pg, which is the average of the LODs using F(ab′)-(PEG)24 beads with only 6E10 or 4G8 (5 pg and 25 pg, respectively). However, the observed LOD was 6-fold higher than expected, which suggests that 6E10 and 4G8 F(ab′) fragments bound to the Aβ1−28 monomer cooperatively rather than independently to increase antigen−antibody affinity. The LOD of Aβ1−28 using 6E10 F(ab′)-(PEG)24 beads was increased 5-fold compared to 6E10 IgG-protein G beads. Meanwhile, in LOD analysis of Aβ1−42, 6E10 F(ab′)-(PEG)24 beads displayed a 4-fold improvement in sensitivity compared with 6E10 IgG-protein G beads (Figure 2B). These results further support that 6E10 F(ab′)-(PEG)24 beads cooperatively bind to Aβ1−28 and Aβ1−42 aggregates. The hetero-F(ab′)-(PEG)24 beads showed higher sensitivity at a 6E10/4G8 ratio of 1:1 compared to 1:3 and 3:1 (w/w) (Table 1). The optimum ratio of 6E10 and 4G8 F(ab′) for MS analysis was determined to be 1:1 (w/w), which supports that they can simultaneously bind one molecule of Aβ1−28. Purification and Detection of Aβ1−28-Spiked Human Plasma. As described above, the enrichment of a target peptide and the removal of nontarget molecules from plasma are efficient approaches for enhancing the detection sensitivity of MALDI-TOF MS. To evaluate the ability of hetero-F(ab′)(PEG)24 beads to purify a peptide of interest from human plasma, we examined the LOD of Aβ1−28 spiked into human plasma using each of the F(ab′)-(PEG)24 beads, F(ab′)-SMCC beads, and IgG-protein G beads. The antibodies noncovalently attached and cross-linked to protein G beads were prepared as for IgG-protein G beads with the possibility of antibody displacement by human IgG during incubation with plasma taken into consideration. When 6E10 F(ab′)-(PEG)24 beads were directly applied to 1 ng of Aβ1−28 spiked into 50 μL of plasma and examined by MALDI-TOF MS, a number of unknown peaks were observed (Figure 3A). Because 6E10 and 4G8 F(ab′) were generated from mouse IgG, antibodies such as human antimouse antibody (HAMA)27 in human plasma can also bind to F(ab′)-(PEG)24 beads. In addition, alkali metal ions from plasma can bind to the negatively charged oxygen atoms in PEG.28 The presence of these substances in plasma could therefore cause the high background noise observed in the mass spectrum. To remove these interferences from MS analysis, preincubation with protein G agarose beads and the matrix additive 2% MDPNA, which is known to eliminate alkali-metal adduct signals,29 were added to the abovementioned processes for MS detection of Aβ1−28 spiked into plasma (Figure 3B). Table 2 shows the LOD of Aβ1−28-spiked human plasma using different types of immunoaffinity beads (F(ab′)-(PEG)24, F(ab′)-SMCC, and cross-linked and noncross-linked IgGprotein G beads). In the IP-MS using noncross-linked IgGprotein G beads, the combination of 6E10 and 4G8 IgG showed higher detection sensitivity than did each IgG alone. We previously reported that symmetroadhesin both with and without a flexible PEG polymer in the hinge region displayed high affinity for antihuman Aβ antibody and provided evidence for the existence of cooperative binding.11 Hence, this result

Supporting Information). This enhanced sensitivity suggests that 6E10 F(ab′) cooperatively binds to aggregated Aβ1−42 due to the PEG linker flexibility. Construction of Hetero-F(ab′)-(PEG)n Beads. Next, we developed hetero-F(ab′)-(PEG)n beads that enable cooperative binding to both amyloid aggregates and monomers. In addition to 6E10, the antihuman Aβ antibody 4G8 that recognizes an epitope at Aβ peptide amino acids 18−22 was used for heteroF(ab′)-(PEG)n beads, because 6E10 and 4G8 reacting with each different epitope had the possibility to bind cooperatively to nonaggregated Aβ peptides. The 4G8 F(ab′)2 fragment was obtained by Lys-C digestion and SEC separation (Frac. 16− 29), which was confirmed by SDS-PAGE under reducing and nonreducing conditions (Figure S-5 in the Supporting Information). 6E10 and 4G8 F(ab′) fragments were then conjugated to magnetic beads through PEG24, which showed higher binding capacity than PEG2 in 1000 μL of TBS-1% OTG (Figure 2A). Binding Ability of Hetero-F(ab′)-(PEG)n Beads. We prepared iso-F(ab′)-(PEG)24 beads obtained by reaction of 150 μg of maleimide-(PEG)24 beads with 0.5 μg of the 6E10 or 4G8 F(ab′) fragment, and hetero-F(ab′)-(PEG)24 beads were obtained by reacting 150 μg of maleimide-(PEG)24 beads with a mixture of 6E10 and 4G8 F(ab′) fragment (0.5 μg total). To optimize the ratio of immobilized 6E10/4G8 F(ab′) for maximum MS sensitivity, 6E10/4G8 ratios of 1:1, 1:3, and 3:1 (w/w) were tested. IgG-protein G beads used as controls were obtained by reacting 150 μg of beads linked to protein G with 0.75 μg of IgG (6E10 or 4G8) or a mixture of 6E10 and 4G8 IgG (1:1, w/w). The binding activity of these beads was evaluated by comparing the LOD of Aβ1−28 with m/z 3264 (Table 1 and Figure S-6 in the Supporting Information), which contains the epitope sequences of 6E10 and 4G8 and exists as a monomer and an aggregate (Figure S-3 in the Supporting Information). 6E10 F(ab′)-(PEG)24 beads had a LOD of 5 pg, demonstrating a 5-fold higher sensitivity than 6E10 IgG-protein Table 1. LODs of Aβ1−28 Recovered by Noncross-linked IgGProtein G and F(ab′)-PEG24 Beads or Pure Aβ1−28 Directly Applied onto MALDI Platesa LOD (S/N ≥ 3) amount in 10 μL solution noncross-linked IgG-protein G 6E10 4G8 6E10/4G8 (1:1 ratio) F(ab′)-PEG24 6E10 4G8 6E10/4G8 (3:1 ratio) 6E10/4G8 (1:1 ratio) 6E10/4G8 (1:3 ratio) directly applied Aβ1−28

(pg)

(fmol)

25 25 25

7.67 7.67 7.67

5 25 5 2.5 5 2.5

1.53 7.67 1.53 0.767 1.53 0.767

Serially diluted Aβ1−28 in 10 μL of TBS-1% OTG was recovered using 150 μg of the different types of immunoaffinity beads, and 1 μL from 10 μL of eluate was applied to MALDI-TOF MS. A volume of 1 μL from 10 μL of serially diluted pure Aβ1‑28 dissolved in 3 mM HCl was directly applied onto the MALDI plate for determination of a reference LOD. The LOD was defined as the amount that yields a measured peak with S/N ≥ 3. a

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also supports that 6E10 and 4G8 IgG can cooperatively bind to the Aβ1−28 monomer by virtue of antibody structural dynamics and that immunoaffinity targeting of multiepitopes is useful for enhancing detection sensitivity. In contrast, the cross-linked IgG-protein G showed lower detection sensitivity than did the noncross-linked IgG-protein G. In addition, the cross-linked IgG-protein G beads with a mixture of 6E10 and 4G8 showed sensitivity that was lower than 4G8 IgG alone. Thus, crosslinking can result in the observed antibody affinity loss caused by conformational changes of the molecule that compensate for the prevention of antibody dissociation.30 The internal crosslinking of antibodies might further induce regulation of the antibody structural dynamics and thereby decrease antibody affinity and the effect of cooperative binding. Among all types of beads tested, 6E10/4G8 F(ab′)-(PEG)24 beads detected the lowest Aβ1−28 concentration (1 pg/50 μL plasma), which was equal to the LOD of pure Aβ1−28 applied directly onto the MALDI plate. By contrast, the LOD of 6E10/4G8 F(ab′)SMCC beads was higher than for 6E10/4G8 F(ab′)-(PEG)24 beads. This result indicated that for enhancing the binding ability of F(ab′) to peptides, PEG, with its flexibility and long spacer length, is preferable to SMCC, which has a short spacer length. From our results, we concluded that multiepitope targeting contributes to high antibody affinity, and moreover, introduction of PEG as a linker enhances this affinity, which leads to higher MS sensitivity. On the other hand, no peaks with S/N ≥ 3 were detected except for Aβ1−28 at a range of m/z 2 000−5 000 for the three types of F(ab′)-(PEG)24 beads, whereas nontarget peaks with S/N ≥ 3 were detected in the tested immunoaffinity beads without PEG, i.e., IgG-protein G beads and F(ab′)-SMCC beads (Figure 4). The use of PEG to introduce flexibility is also effective in reducing nonspecific binding because of its hydrophilicity,31 which could contribute to the high specificity of Aβ1−28 isolation as demonstrated in Figure 4. Detection of Aβ1−40 from Human Plasma. To detect endogenous Aβ peptides in human plasma, 250 μL of healthy human plasma was diluted to 5 mL in TBS-1% OTG and subsequently immunoprecipitated using 750 μg of 6E10/4G8 F(ab′)-(PEG)24 beads (1:1, w/w). The purified peptides were then analyzed by MALDI-TOF MS (Figure 5A). The m/z value of the observed peak indicated that it corresponds to the amino acid sequence of human Aβ1−40 (m/z 4330). To verify the detected peak at m/z 4330, a MS/MS spectrum was obtained using collision induced dissociation (CID; Figure 5B). A number of product ions observed corresponded to the fragment peptides from human Aβ1−40, especially those generated by preferential cleavage at the C-termini aspartic acid or glutamic acid residues, and a Mascot score of 26 was obtained. The Aβ1−40 concentration was determined to be 51.7 pM in the tested plasma using ELISA. These results demonstrated that the detection of endogenous plasma Aβ1−40 in MALDI-TOF MS was achieved through IP using 6E10/4G8 F(ab′)-(PEG)24 beads, although other molecules could be present at m/z 4330 owing to the generation of unknown product ions.

Figure 3. MALDI-TOF mass spectra of Aβ1−28 (m/z 3 264) purified by 6E10 F(ab′)-(PEG)24 beads. (A) Immunoprecipitation of human plasma spiked with 1 ng of Aβ1−28 using 6E10 F(ab′)-(PEG)24 beads, which was followed by MALDI-TOF MS measurement. (B) Same as in part A with pretreatment of the sample preparation with protein G agarose beads and the use of MDPNA as an additive to the CHCA matrix.

Table 2. LODs of Aβ1−28 Purified from Aβ1−28-Spiked Plasma by Immunoaffinity Beads, or Pure Aβ1−28 Directly Applied onto MALDI Platesa LOD (S/N ≥ 3) amount spiked into 50 μL of plasma (pg) noncross-linked IgG-protein 6E10 50 4G8 25 6E10/4G8 5 (1:1 ratio) cross-linked IgG-protein G 6E10 500 4G8 50 6E10/4G8 100 (1:1 ratio) F(ab′)-PEG24 6E10 2.5 4G8 10 6E10/4G8 1 (1:1 ratio) F(ab′)-SMCC 6E10/4G8 50 (1:1 ratio)

concn in spiked-plasma

concn after dilution of spiked-plasma

(fmol)

(pM)

(pM)

G 15.3 7.67 1.53

306 153 30.6

15.3 7.67 1.53

153 15.3 30.7

3060 306 614

153 15.3 30.7

0.767 3.07 0.307

15.3 61.4 6.14

0.767 3.07 0.307

15.3

306

15.3

amount in 10 μL of soln directly applied Aβ1−28

(pg)

(fmol)

1

0.307

Serially diluted Aβ1−28 was spiked into 50 μL of healthy human plasma, and the spiked plasma was diluted to 1 mL in TBS-1% OTG to obtain a 20-fold dilution. After immunoprecipitation using 150 μg of each different type of immunoaffinity beads, 1 μL from 10 μL of eluate was applied to MALDI-TOF MS. A volume of 1 μL from 10 μL of serially diluted pure Aβ1−28 dissolved in 3 mM HCl was directly applied onto the MALDI plate for determination of a reference LOD. The LOD was defined as the amount that yields a measured peak with S/N ≥ 3. a

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CONCLUSIONS We developed hetero-F(ab′)-(PEG)24 beads with F(ab′) fragments from 6E10 and 4G8 to design a purification approach capable of producing a high purification efficiency that will enhance MS sensitivity. 6E10/4G8-F(ab′)-(PEG)24 beads achieved a sensitivity of 307 amol of Aβ1−28/50 μL plasma, corresponding to 6.14 pM. This LOD represents a 25− F

dx.doi.org/10.1021/ac303344h | Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 5. MS detection and MS/MS analysis of endogenous Aβ1−40 in human plasma. (A) The spectrum shows an Aβ1−40 peak detected by MALDI-TOF MS after immunoprecipitation of 250 μL of human plasma using 6E10/4G8 F(ab′)-(PEG)24 beads. (B) The MS/MS spectrum for the precursor ion of Aβ1−40 at m/z 4330 was acquired using CID to verify the identity of the peptide. Peak from an internal fragmentation is indicated with “I”. Peak labeled with an asterisk corresponds to the background peaks in the negative control with 15 mM HCl.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 4. MALDI-TOF mass spectra of detection limits for Aβ1−28spiked human plasma. Each spectrum shows the peaks with S/N ≥ 3 in the lowest LOD of Aβ1−28 spiked into 50 μL of plasma using each type of bead (noncross-linked 6E10/4G8 IgG-protein G (A), crosslinked 4G8 IgG-protein G (B), 6E10/4G8 F(ab′)-PEG24 (C), and 6E10/4G8 F(ab′)-SMCC (D)). Each Aβ1−28 concentration in these spectra corresponds to the detection limit shown in Table 2.

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

*Phone: +81-75-823-2897. E-mail: [email protected]. Notes

50-fold improvement in sensitivity compared to conventional IP using noncross-linked 6E10 or 4G8 IgG-protein G magnetic beads. Our study suggests that two different kinds of F(ab′) fragments linked to beads via PEG can have greater flexibility and promote cooperative binding, which in turn produces enhanced antibody affinity. Furthermore, compared to conventional IP using protein G, highly specific MS measurement of peptides in human plasma can be achieved owing to the exclusion of nontarget molecules by hetero-F(ab′)-(PEG)24 beads. Finally, IP using 6E10/4G8-F(ab′)-(PEG)24 beads was successfully applied to MS detection of endogenous Aβ1−40 in healthy human plasma. To our knowledge, this is the first study to detect endogenous plasma Aβ1−40 by MALDI-TOF MS. Because Aβ1−42 is known to be present at lower concentrations than Aβ1−40 in human plasma, further improvement of our IPMS method is needed for the detection of other Aβ isoforms, including Aβ1−42. Although the sensitivity of different peptides could depend on their molecular weight and hydrophobicity, the hetero-F(ab′)-(PEG)24 bead technology has the potential to allow analysis of previously undetected peptides, such as plasma Aβ1−40 that are present in peripheral blood.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by a grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). We thank Dr. Kazuhisa Konishi for helpful comments in the preparation of the manuscript.

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REFERENCES

(1) Zhu, Q.; Kasim, A.; Valkenborg, D.; Burzykowski, T. Int. J. Proteomics 2011, 928391. (2) Young, S. A.; Julka, S.; Bartley, G.; Gilbert, J. R.; Wendelburg, B. M.; Hung, S.-C.; Anderson, W. H. K.; Yokoyama, W. H. Anal. Chem. 2009, 81, 9120−9128. (3) Wolf, R.; Fred, R.; Hoffmann, T.; Demuth, H.-U. J. Chromatogr., A 2001, 926, 21−27. (4) Wolf, R.; Hoffmann, T.; Fred, R.; Demuth, H.-U. J. Chromatogr., B 2004, 803, 91−99.

G

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(5) Hawkridge, A. M.; Heublein, D. M.; Bergen, H. R.; Cataliotti, A.; Burnett, J. C.; Muddiman, D. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17442−17447. (6) Lopez, M. F.; Rezai, T.; Sarracino, D. A.; Prakash, A.; Krastins, B.; Athanas, M.; Singh, R. J.; Barnidge, D. R.; Oran, P.; Borges, C.; Nelson, R. W. Clin. Chem. 2010, 56, 281−290. (7) Niederkofler, E. E.; Kiernan, U. A.; O’Rear, J.; Menon, S.; Saghir, S.; Protter, A. A.; Nelson, R. W.; Schellenberger, U. Circ.: Heart Failure 2008, 1, 258−264. (8) Wang, Y.; Keck, Z.-Y.; Saha, A.; Xia, J.; Conrad, F.; Lou, J.; Eckart, M.; Marks, J. D.; Foung, S. K. H. J. Biol. Chem. 2011, 286, 44218−44233. (9) Kim, H. S.; Lo, S.-C.; Wear, D. J.; Stojadinovic, A.; Weina, P. J.; Izadjoo, M. J. J. Immunol. Methods 2011, 372, 146−161. (10) Lippow, S. M.; Wittrup, K. D.; Tidor, B. Nat. Biotechnol. 2007, 25, 1171−1176. (11) Capon, D. J.; Kaneko, N.; Yoshimori, T.; Shimada, T.; Wurm, F. M.; Hwang, P. K.; Tong, X.; Adams, S. A.; Simmons, G.; Sato, T.-A.; Tanaka, K. Proc. Jpn. Acad. Ser. B 2011, 87, 603−616. (12) Bartolini, M.; Naldi, M.; Fiori, J.; Valle, F.; Biscarini, F.; Nicolau, D. V; Andrisano, V. Anal. Biochem. 2011, 414, 215−225. (13) Friedrich, R. P.; Tepper, K.; Rö nicke, R.; Soom, M.; Westermann, M.; Reymann, K.; Kaether, C.; Fändrich, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1942−1947. (14) Gouras, G. K.; Almeida, C. G.; Takahashi, R. H. Neurobiol. Aging 2005, 26, 1235−44. (15) Blennow, K. NeuroRx 2004, 1, 213−225. (16) Luis, C. A.; Abdullah, L.; Ait-Ghezala, G.; Mouzon, B.; Keegan, A. P.; Crawford, F.; Mullan, M. Int. J. Alzheimers Dis. 2011, 2011, 786264. (17) Portelius, E.; Brinkmalm, G.; Tran, A. J.; Zetterberg, H.; Westman-Brinkmalm, A.; Blennow, K. Neurodegener. Dis. 2009, 6, 87− 94. (18) Portelius, E.; Westman-Brinkmalm, A.; Zetterberg, H.; Blennow, K. J. Proteome Res. 2006, 5, 1010−6. (19) Gelfanova, V.; Higgs, R. E.; Dean, R. A.; Holtzman, D. M.; Farlow, M. R.; Siemers, E. R.; Boodhoo, A.; Qian, Y.-W.; He, X.; Jin, Z.; Fisher, D. L.; Cox, K. L.; Hale, J. E. Brief. Funct. Genomics Proteomics 2007, 6, 149−158. (20) 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. (21) Mehta, P. D.; Pirttilä, T.; Mehta, S. P.; Sersen, E. A.; Aisen, P. S.; Wisniewski, H. M. Arch. Neurol. 2000, 57, 100−105. (22) Le Bastard, N.; Aerts, L.; Leurs, J.; Blomme, W.; De Deyn, P. P.; Engelborghs, S. Neurochem. Int. 2009, 55, 820−825. (23) Dimeski, G.; Barnett, R. J. Crit. Care Resuscitation J. Australas. Acad. Crit. Care Med. 2005, 7, 12−15. (24) Hu, S.; Loo, J. A.; Wong, D. T. Proteomics 2006, 6, 6326−6353. (25) Cerf, E.; Gustot, A.; Goormaghtigh, E.; Ruysschaert, J.-M.; Raussens, V. FASEB J. Off. Publ. Federation Am. Soc. Exp. Biol. 2011, 25, 1585−1595. (26) Shimada, T.; Toyama, A.; Aoki, C.; Aoki, Y.; Tanaka, K.; Sato, T.-A. Rapid Commun. Mass Spectrom. 2011, 25, 3521−3526. (27) Kricka, L. J. Clin. Chem. 1999, 45, 942−956. (28) Kuki, Á .; Nagy, L.; Shemirani, G.; Memboeuf, A.; Drahos, L.; Vékey, K.; Zsuga, M.; Kéki, S. Rapid Commun. Mass Spectrom. 2012, 26, 304−308. (29) Kuyama, H.; Sonomura, K.; Nishimura, O. Rapid Commun. Mass Spectrom. 2008, 1109−1116. (30) Sousa, M. M.; Steen, K. W.; Hagen, L.; Slupphaug, G. Proteome Sci. 2011, 9, 45. (31) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Adv. Drug Delivery Rev. 2003, 55, 403−419.

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