A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures.

Special Issue Article Received: 03 December 2013,

Revised: 27 February 2014,

Accepted: 13 March 2014,

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/jmr.2375

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures† Mahmoud Al-Majdouba‡, Kwabena F. M. Opunia‡, Yelena Yefremovaa, Cornelia Koya, Peter Lorenzb, Reham F. El-Kasedc, Hans-Jürgen Thiesenb and Michael O. Glockera* The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity-adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody–epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)-containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody–epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His-tag-containing recombinant human glucose-6-phosphate isomerase in combination with an antiHis-tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody–epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto-antigen in combination with a rabbit polyclonal anti-TRIM21 antibody. Peptide chip analysis showed that antibody–epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA-containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody–antigen complexes under neutral pH and low salt conditions for subsequent investigations. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: affinity mass spectrometry; mass spectrometric epitope mapping; antibody-based affinity chromatography; protein A; protein G´; antibody–epitope complexes

* Correspondence to: M. O. Glocker, Proteome Center Rostock, Medical Center and Natural Science Faculty, University of Rostock, Schillingallee 69, P.O. Box 100 888, 18055 Rostock, Germany. E-mail: [email protected]

INTRODUCTION

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Autoimmune diseases constitute a major challenge and difficulty in clinical diagnosis because the presence of autoantibodies occurs in healthy individuals to some extent as well. Further, autoantibodies against the same autoantigen can be found in patients suffering from different autoimmune diseases, impeding clear disease diagnostics on the antibody–antigen level (Jacobson et al., 1997; Scofield, 2004; Selmi, 2010). Disease-specific epitope peptides have been suggested to be used instead of full-length antigens to overcome current diagnostic limitations, and specialized arrays with peptides/epitopes of importance for a given disease (disease-specific arrays) are desired in the clinic for patient screening and/or disease diagnostics (Andresen and Grotzinger, 2009). Consequently, epitope mapping and detailed characterization of binding properties have become important for designing the so-called next generation chip arrays (Al-Majdoub et al., 2013a; Al-Majdoub et al., 2013b). To fulfill these tasks, fast and miniaturized isolation and preparation procedures are needed to gain access to intact antibody–epitope complexes.

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†

‡

This article is published in Journal of Molecular Recognition as part of the Special Issue ‘State-of-the-art B-cell Epitope Discovery’, edited by Dr Yasmina Abdiche, Dr Arvind Sivasubramanian, Dr Jerry Slootstra, Dr Darren Flower and Professor Edouard Nice. Both authors contributed equally to the manuscript

a M. Al-Majdoub, K. F. M. Opuni, Y. Yefremova, C. Koy, M. O. Glocker Proteome Center Rostock, University Medicine Rostock, Rostock, Germany b P. Lorenz, H.-J. Thiesen Institute of Immunology, University Medicine Rostock, Rostock, Germany c R. F. El-Kased Microbiology and Immunology Faculty of Pharmacy, The British University in Egypt, Cairo, Egypt Abbreviations: SEC, size exclusion chromatography; rhGPI, recombinant human glucose-6-phosphate isomerase; rhTRIM21, recombinant human tripartite motif 21; Fc, fragment of crystallization; IPA, indole propionic acid; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; MOPS, 3-(N-morpholino)propanesulfonic acid; DHB, dihydroxybenzoic acid; ACN, acetonitrile; TFA, trifluroacetic acid.

Copyright © 2014 John Wiley & Sons, Ltd.

ISOLATION OF ANTIBODY–EPITOPE COMPLEXES

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MATERIALS AND METHODS Recombinant human proteins Details on the sequence and production of recombinant human tripartite motif 21 (rhTRIM21) and recombinant human glucose6-phosphate isomerase (rhGPI) protein have been described (Al-Majdoub et al., 2013a; Al-Majdoub et al., 2013b). Each protein (100 μg; 1 μg/μl, dissolved in 50 mM sodium phosphate, 1000 mM sodium chloride, and 8 M urea, pH 7.4) was reduced with 10 μl of freshly prepared 10 mM dithiothreitol in 100 mM ammonium bicarbonate solution and incubated at 56°C for 30 min, despite the fact that heating results in (partial) carbamylation of amino groups. Subsequent alkylation was achieved by addition of 20 μl freshly prepared 55 mM iodoacetamide solution in 100 mM ammonium bicarbonate and incubation at room temperature in the dark for 30 min. The proteins were then precipitated using four volumes of methanol, one volume of chloroform, and three volumes of water with respect to the protein sample volume. The precipitates were resolublized in 6 M urea/2 M thiourea dissolved in water, producing final protein concentrations of 1 μg/μl each. Trace amount of salts from the previous work-up steps may still be present. Lys-C in-solution digestion of antigens For in-solution digestion of proteins, endoproteinase Lys-C (Roche Diagnostics GmbH, Mannheim, Germany; reconstituted according to the manufacturer’s protocol) was used in enzyme to substrate ratios of 1:50. Digestion was performed at room temperature over night. The samples were frozen at 20°C to stop the digestion. ZipTip C18 tips (Millipore, Billerica, USA) were used as previously described (Al-Majdoub et al., 2013b) to desalt 5 μl of the resulting peptide solutions. Elution solvents were removed using a SpeedVac concentrator. Peptides were resolubilized in 5 μl of either 100 mM ammonium bicarbonate or of 200 mM ammonium acetate. Formation of immune complexes The epitope extraction experiments were performed using peptide mixtures of digested antigens and either the mouse monoclonal anti-His-tag antibody (AbD Serotec MCA (clone AD1.1.20), Düsseldorf, Germany; this antibody works in the applications Western blot, ELISA, and immunoprecipitation, and at least 12 peer-reviewed publications made use of it) or the rabbit polyclonal anti-TRIM21 antibody (sc-20960, H-140, lot 0503; Santa Cruz Biotechnology, Inc., Heidelberg, Germany). Antibodies (0.5 pmol/μl) were dissolved in 10 mM phosphate buffered saline, pH 7.4. Each epitope extraction experiment was performed using a programmable Rainin E4 XLS eightchannel electronic pipette (Mettler Toledo, Germany), equipped with 200 μl PureSpeed Protein Tips containing 5 μl protein G´ or protein A resin (Mettler Toledo, Germany) (Al-Majdoub et al., 2013b). Note that protein G´ is the name of the recombinantly engineered protein (Goward et al., 1990). This term was introduced to indicate sequential differences of the commercialized product with respect to the bacterial wild-type protein. Protein A has been reported to better bind to mouse antibodies, whereas antibodies from human and rabbit origin bind better to protein G (Guss et al., 1986). Different binding strengths were also reported to be dependent on the antibody isotype.


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Antibody-based affinity chromatography has been amply applied for antigen purification and detailed antibody–antigen binding analyses, respectively. Supporting materials, such as Sepharose beads (Suckau et al., 1990; Papac et al., 1994; Hochleitner et al., 2000; Griffiths et al., 2011) and magnetic beads (Lu et al., 1998; Jankovicova et al., 2008), are available for covalent attachment of antibodies, through which the specificity of an affinity chromatography system is provided. Alternatively, noncovalent immobilization of antibodies on protein A and/or protein G´ in columns/pipette tips (Zhao and Chait, 1994; AlMajdoub et al., 2013b) or by immunoprecipitation (Ansong et al., 2006) has been applied successfully for antibody–antigen or epitope mapping studies. The advantages of noncovalent antibody binding to either protein A or protein G´ over covalent attachment of the antibody on a support material are as follows: (i) binding through the fragment of crystallization (Fc) or fragment of antigen binding (Fab) domains does not require chemical reactions that may result in unwanted modifications; (ii) paratope exposure to the solvent is guaranteed; and (iii) antibody attachment is reversible (Reid et al., 2010). With covalent attachment of the antibody to a support material, the release of the binding partner (antigen) seems not delicate, inviting the application of rather crude elution conditions. Denaturing high salt elution buffers (mostly molar concentrations) have been applied almost indiscriminately including both acidic and basic conditions (Tsang and Wilkins, 1991; Ben-David and Firer, 1996). They lead to efficient dissociation of the antibody–antigen complex presumably by destroying the tertiary structures of the proteins, that is, the antigen and the antibody (Fornsted, 1984). This elution principle has been applied for mass spectrometric epitope mapping (Suckau et al., 1990; Macht et al., 1996; Hager-Braun et al., 2006; Stefanescu et al., 2011) and related applications (Stefanescu et al., 2007; Popescu et al., 2008; Jimenez-Castells et al., 2012). Nevertheless, depending on the complexity of the antigen-containing solution, for example, patient serum, the eluted target molecules (antigens) are usually accompanied by unspecifically bound “background” molecules that arise from nonspecific interactions of (unknown) molecules with the bead surfaces and those of the capturing molecules, respectively. Because of lack of control during elution, both the target molecule and the “background” are released simultaneously, making it difficult if not impossible to decide between specific and unspecific binding without employing suitable controls. By contrast, an affinity system with a reversibly bound antibody demands to pick and choose carefully for appropriate elution conditions right from the beginning. In the ideal case, after having bound the antigen (and washing), the reversibly attached antibody–antigen complex can be released without interfering with the paratope–epitope interaction, thereby providing the intact immune complex for further analyses. Here, we present an affinity chromatography system that provides specificity both during antibody–antigen complex formation and release of intact immune complexes by “soft” elution. The system consists of protein A and/or protein G´ resins that are embedded in pipette tips (Al-Majdoub et al., 2013b). Indole propionic acid (IPA)-containing buffers were found to be well functioning as “soft” eluents. Successful elution of intact immune complexes when using this novel isolation strategy was proven by size exclusion chromatography (SEC) in combination with mass spectrometry (MS).

M. AL-MAJDOUB ET AL. Equilibration of the PureSpeed Protein Tips was done using one cycle for aspiration/dispension of 100 μl of equilibration buffer (10 mM sodium dihydrogen phosphate/140 mM sodium chloride, pH 7.4). Mouse monoclonal anti-His-tag antibody and rabbit polyclonal anti-TRIM21 antibody-containing buffers (50 μl), respectively, were used for immobilizing the antibodies on the protein A and protein G´ resins using six cycles of aspiration/ dispension. Next, 50 μl of diluted rhGPI or rhTRIM21 (1 pmol/μl) peptide mixtures with molar ratios of 2:1 (antigen to antibody) was allowed to flow through the column for six successive cycles over a period of 10 min to allow the antibody–peptide binding. This was followed by three washing steps (three cycles each) using for each cycle 100 μl of wash buffer 1 (10 mM sodium dihydrogen phosphate/140 mM sodium chloride in water, pH 7.4), wash buffer 2 (140 mM sodium chloride in water, pH 7.4), and wash buffer 3 (14 mM sodium chloride in water, pH 7.4). Then, elution of the antibody plus epitope peptide was performed either with 20 μl IPA-solution (50 mM indole-3propionic acid, Sigma-Aldrich Chemie GmbH, Steinheim, Germany, in 10% ethanol/100 mM ammonium bicarbonate, pH 7.0) using six cycles or with 20 μl sodium dihydrogen phosphate solution (200 mM sodium dihydrogen phosphate/140 mM sodium chloride in water, pH 2.5). Eluted solvents were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption ionization MS (MALDI-MS), and SEC, respectively.

voltages (20.0 kV) linear mode; (23.0 kV) reflector mode were set. Acquired spectra were analyzed using FlexAnalysis 2.4 and BioTools 3.0 programs (Bruker Daltonik, Bremen, Germany). Native MS TRIM21 polyclonal antibody (5 μl of 0.2 μg/μl) was buffer exchanged into 200 mM ammonium acetate, pH 7.1, using a centrifugal filter (Amicon Ultra with cutoff 50 K; Millipore Corporation, Ireland) for concentration and rebuffering. Lys-Cderived rhTRIM21 peptide solution (1 μl) in 200 mM ammonium acetate, pH 7.3, was added to the anti-TRIM21 solution and reached a molar ratio of 10:1. The mixture was loaded into EconoTipTM emitters (ECONO10, New Objective Inc., Woburn, MA, USA) using microloader pipette tips (Eppendorf, Hamburg, Germany). Off-line nanoelectrospray ionization (nano-ESI) MS was performed on a quadrupole-ToF II (Q-ToF II) instrument (Waters MS-Technologies, Manchester, UK) and calibrated externally using cesium iodide solutions. The pressure conditions within the mass spectrometer were adjusted to preserve noncovalent interactions as previously described (Sobott et al., 2002). The following instrumental settings were used: positive ion mode; source temperature, 40° C; capillary voltage, 1.7 kV; cone gas, 100 l/h; sample cone, 180 V; extractor cone, 20 V; collision energy, 4 V; quadrupole analyzer pressure, 2.20 × 10 3 mbar; and Tof analyzer pressure, 3.66 × 10 6 mbar. Spectra were accumulated for approximately 10 min. The MassLynx software (Micromass, Manchester, UK) was used for data acquisition and processing.

SDS-PAGE analysis Each eluted solvent (10 μl; antibody plus peptide(s)) was mixed with 2.5 μl of five-fold SDS sample buffer (156 mM tris (hydroxymethyl)aminomethane (TRIS), 5% SDS, 25% glycerol, and bromophenol blue) and loaded onto a NuPAGE Novex 12% Bis–TRIS gel (Invitrogen/Thermo Fisher Scientific, Germany). The gel was placed into an Xcell SurelockTM MiniCell (Invitrogen, Karlsruhe, Germany) electrophoresis chamber, and voltage was set to 200 V for 65 min using 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (25 mM MOPS, 25 mM TRIS, 3.5 mM SDS, 1 mM ethylenediaminetetraacetic acid) as a running buffer. The Broad Range Marker (New England BioLabs, Frankfurt/Main, Germany) was used as an apparent molecular mass standard. (Al-Majdoub et al., 2013a)

SEC SEC columns were constructed as described (El-Kased et al., 2011; open access paper: http://www.omicsonline.org/0974276X/JPB-04-001.pdf). The SEC columns were washed with 100 μl of water followed by 100 μl of 50 mM ammonium bicarbonate (pH 8.0). Eluted samples (10 μl each; antibody plus peptide(s)) were loaded and allowed to stand for 20 min. Then, 10 μl of elution buffer (50 mM ammonium bicarbonate) was added repeatedly, and solvents were pushed through the columns with the help of an air-filled syringe for fractionation. Up to 24 fractions of about 5 μl each were collected. All fractions were desalted using ZipTip C18 tips (Millipore, Billerica, USA), and 3 μl each of the resulting peptide samples, dissolved in 80% ACN/1% TFA solution, was used for MS analyses.

MALDI time of flight (ToF) MS analysis

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Eluted samples (antibody plus peptide(s); 2 μl each) were submitted for desalting with ZipTip C18 tips (Millipore, Billerica, USA). Desalted peptide mixtures (2 μl) were spotted onto an AnchorChipTM 600/384 target plate (Bruker Daltonik, Bremen, Germany), and for spectra calibration, the standard peptide mixture (Al-Majdoub et al., 2013b) was used. Next, 1.0 μl of 2,5dihydroxybenzoic acid (DHB, LaserBio Labs, France) matrix solution (5 mg DHB dissolved in 1000 μl of 50% acetonitrile (ACN)/0.1% trifluroacetic acid (TFA)) was added to spotted samples and calibration peptide mixture, respectively, and mixed on the target. The spotted samples were air dried at room temperature and inserted into the SCOUT source of the Reflex III MALDI ToF MS instrument (Bruker Daltonik, Bremen, Germany) (Sinz et al., 2002). All data were acquired in positive ion reflector mode (400 shots per spectrum) in the mass range of m/z 680–4000. A nitrogen laser (wavelength 337 nm, pulse width 3–5 ns) was used for analyte desorption. Acceleration


Peptide chip analysis Multiparallel epitope mapping was done with four RepliTopeTM peptide microarrays (JPT Peptide Technologies GmbH, Berlin, Germany) as described (Al-Majdoub et al., 2013a) with the following modifications. The arrays were stained with a primary antibody (rabbit pAb anti-TRIM21; Santa Cruz: sc 20960) in a 1:100 dilution with TRIS-buffered saline (TBS) buffer (TBS, 0.05% Tween 20, 0.1% bovine serum albumin buffer) and incubated at room temperature for 4 h (100 μl with a final concentration of 2 μg/ml). Following the usual four washing steps (5-min incubations each) with TBS buffer, the microarray slides were either incubated at room temperature for 10 min under agitation using 25 ml of glycine buffer (0.1 M glycine/HCl, pH 2.8) or with 25 ml IPA-buffer (50 mM IPA dissolved in 100 mM ammonium bicarbonate containing 10% ethanol, pH 7.0) or with another round of 25 ml TBS-buffer (used as a positive control). Subsequently, the microarrays were once rinsed with 25 ml of


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ISOLATION OF ANTIBODY–EPITOPE COMPLEXES the respective buffer in which they were incubated in before. The fourth array was probed with TBS buffer instead of the primary antibody and washed with TBS buffer only (background control). After these steps, the chips were processed as usual; that is, they were incubated with a secondary antibody (Zenon goat anti-rabbit IgG-AlexaFluor-657, Invitrogen Z25308; used at 0.8 μg/ml final concentration) at room temperature for 2 h. Finally, chips were washed as described, scanned at 10 μm resolution as described previously (Lorenz et al., 2009). Data analysis followed the in-house developed work flow (Hecker et al., 2012).

Nano-ESI MS Off-line nano-ESI-MS was performed with protein G´ using a Waters Q-ToF II instrument (Waters MS-Technologies, Manchester, UK). Protein G´ (0.5 μg/μl) was dissolved in the following: (i) 2% aqueous acetic acid : MeOH (95:5 v/v), pH = 2.6; (ii) 50 mM aqueous ammonium acetate : MeOH (95:5 v/v), pH = 6.8; and (iii) 25 mM IPA in 100 mM aqueous ammonium bicarbonate : EtOH : MeOH (90:5:5 v/v/v), pH 7.5, respectively. Each of these solutions (5 μl) was loaded into EconoTipTM emitters (ECONO10, New Objective Inc., Woburn, MA, USA) using microloader pipette tips (Eppendorf, Hamburg, Germany). The needle potential was adjusted to 2.0 kV. The cone voltage was set to 80 V for solutions with neutral pH and to 40 V for acidic solvents. Extractor was set to 3 V, rangefinder lens to 1.2 V. A nitrogen counterflow was set to 50 l/h to assist desolvation. The ion–spray interface temperature was 60–70ºC for all measurements. A scan rate of 7 s/scan was used for spectrum recording, and spectra were acquired with a digitization rate of 4 GHz using a mass window of m/z 800–4000 and microchannel plate was set to 1950 V. Data acquisition and processing was performed with the MassLynx software version 4.1.

Figure 1. Work flow of the antibody–epitope extraction procedure using protein A or protein G´ columns with neutral and acidic elution conditions, respectively, and with subsequent SEC, SDS-PAGE, and MALDI-MS analysis.

RESULTS Affinity chromatography with reversibly immobilized antibodies

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Figure 2. SDS-PAGE analysis of the mixtures that contained antibody and peptide(s) eluted from protein A and protein G´, respectively, under neutral conditions. M: broad range mass marker. Apparent molecular masses are given in kDa. Lane 1: anti-His-tag antibody plus peptide mixture after LysC digestion of rhGPI. Lane 2: anti-His-tag antibody (positive control). Lane 3: anti-TRIM21 antibody plus peptide mixture after LysC digestion of rhTRIM21. Lane 4: anti-TRIM21 antibody (positive control).

The experimental setup was extended by using a rabbit polyclonal anti-TRIM21 antibody and the peptide mixture derived from Lys-C-digested rhTRIM21. Only this time, the antibody was noncovalently immobilized on protein G´ in 200 μl PureSpeed Protein Tips. SDS-PAGE analysis showed that the anti-TRIM21 antibody was also effectively eluted under neutral conditions (Figure 2, lane 3) using the IPA solution. MALDI-ToF-MS analysis of the eluted sample showed the presence of the epitope peptide (QK232NFLVEEEQRQLQELEKDEREQLRILGEK260EA) that is recognized by the anti-TRIM21 rabbit polyclonal antibody via its “L-E-Q-L” binding motif, through detecting its ion signal at



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The “soft” antibody–epitope extraction procedure under neutral pH elution conditions (Figure 1) was developed using the mouse monoclonal anti-His-tag antibody and the His-tag-containing peptide mixture derived from Lys-C-digested rhGPI. First, the antibody was noncovalently immobilized on protein A in 200 μl PureSpeed Protein Tips. Then, the antigen peptide mixture from the digest was added to generate the antibody–epitope complex under slightly alkaline conditions (pH 8). After washing off unbound peptides, the epitope–antibody complex constituents were eluted with neutral buffers (50 mM IPA solution, pH 7.0). IPA (predominantly because of its structural similarity to tryptophan) should be able to interfere with aromatic amino acid residues of protein G´, thereby opening up the interaction surface with the antibody. Eluents were analyzed by SDS-PAGE in which the eluted antibody was detected as a band at 150 kDa (Figure 2, lane 1) and by MALDI-MS, in which the epitope peptide, here, the His-tag-containing peptide of rhGPI (IK549QQREARVQLEHHHHHH565), was detected at m/z 2079.09 (Figure 3A). Subsequent elution with an acidic solvent (pH 2.5) and analysis of the eluent by SDS-PAGE and MALDI-MS showed that neither the antigen peptide nor the antibody had remained on the column (data not shown).

M. AL-MAJDOUB ET AL.

Figure 4. SDS-PAGE analysis of the SEC fractions that contained immune complexes consisting of rhTRIM21 epitope peptides and antiTRIM21 antibody that eluted from protein G´ under neutral conditions. Each numbered lane contains consecutive SEC fractions. Lane M: molecular mass marker. Lane C: anti-TRIM21 antibody (positive control).

Figure 3. MALDI-ToF-MS analysis of the mixtures that contained antibody and peptide(s) eluted from protein A and protein G´, respectively, under neutral conditions. (A) His-tag-containing peptide (ion signal at m/z 2079.03) of rhGPI (cf. Lane 1 in Figure 2). (B) Epitope peptide (ion signal at m/z 3498.85) of rhTRIM21 (cf. Lane 3 in Figure 2). Black dots label unidentified ion signals, asterisks point to loss of water, and triangles indicate carbamylation products. DHB was used as matrix. The identity of the epitope peptides was confirmed by MS/MS fragmentation (Al-Majdoub et al., 2013b), but the peptide ion signals marked with dots were not analyzed further as these were already addressed as background signals by differential analysis with unrelated control antibodies in previous studies. Inserts show magnifications of the epitope peptide ion signals.

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m/z 3498.85 (Figure 3B). Note that the antibody seems quite well discriminating between the “assembled” α-helical epitope motif “L-E-Q-L” with respect to a sequential “LQEL” motif. The different binding strength between the two types of motifs was earlier determined by peptide chip analysis (Al-Majdoub et al., 2013a). An associated ion signal at m/z 3541.81 arose from carbamylation (marked with a triangle in Figure 3B). According to molecular modeling of the epitope peptide and proven by experimental data (Al-Majdoub et al., 2013b), the epitope peptide assumes an α-helical secondary structure. The potential carbamylation sites (N-terminal amino acid and Lys248 (but not Lys260)) are not part of the secondary structure hydrogen bonds that are needed for forming the α-helix (cf. Supplemental Figure 1). Also, the mentioned amino groups are not involved in the antibody-binding surface (provided by the “L-E-Q-L” motif). Hence, carbamylation of any of the concerned residues is not of hindrance for antibody binding in this case. As controls, we performed the epitope extraction experiments with both the anti-His-tag antibody plus the rhGPI peptide mixture and the anti-TRIM21 antibody plus the rhTRIM21 peptide


Figure 5. MALDI-ToF-MS analysis of the SEC fractions that contained immune complexes consisting of rhTRIM21 epitope peptides and antiTRIM21 antibody and/or unbound peptides upon elution from protein G´ under neutral (left) and acidic (right) conditions, respectively. SEC fraction numbers are given at the right.

mixture, however, by changing to acidic elution conditions (pH 2.5). SDS-PAGE analysis showed that antibodies were found in the eluents (Supplemental Figure 2, lanes 1 and 3) as expected. MALDI-ToF-MS analysis (Supplemental Figure 3) of the eluted samples showed that they contained the epitope peptide ion signals at either m/z 2079.08 (His-tag epitope from rhGPI) or at m/z 3498.86 (rhTRIM21 epitope peptide) as well. Characterization of complex components by SEC SEC was used to test whether the antibody–epitope constituents that were obtained in the eluents under neutral pH conditions were present as intact noncovalent complexes or as dissociated components. This distinction was possible because of our miniaturized SEC system in which large molecules, such as antibodies and/or antibody–epitope peptide complexes, are eluted early (typically in fractions 2 to 6; Figure 4), whereas small compounds, such as peptides, are eluted late (typically in fractions 12 to 17). All fractions (5 μl each) were subjected to MALDI-MS analysis by which the epitope peptides could be observed with high sensitivity when present (Figure 5). MALDI-ToF-MS analysis of SEC fractions from the rhTRIM21 epitope extraction experiments showed the epitope peptide (corresponding ion signal at m/z 3498.83) in the early fractions


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ISOLATION OF ANTIBODY–EPITOPE COMPLEXES (Figure 5 and Supplemental Figure 4) after neutral elution and its absence in the late SEC fractions. As expected, MALDI-ToF-MS analysis of SEC fractions from the sample eluted under acidic conditions showed the corresponding ion signal at m/z 3498.86 of the rhTRIM21 epitope peptide in the late fractions, whereas it was not present in the early fractions. Comparably, MALDI-ToF-MS analysis of SEC fractions obtained with anti-His-tag mouse monoclonal antibody and rhGPI peptides upon elution under neutral pH conditions showed the corresponding ion signal at m/z 2079.05 in the early fractions (Supplemental Figure 5), whereas this ion signal was not present in the late fractions. By contrast, MALDI-ToF-MS analysis of SEC fractions from the eluted sample using acidic conditions detected the His-tag epitope peptide from rhGPI in the late fractions and its absence in the early fractions. These results are consistent with the presence of an intact antibody–epitope peptide complex during neutral elution from the protein G´ and the protein A column, respectively. Complexes were only dissociated during the MALDI matrix preparation procedure on the target.

Peptide chip analysis of antibody–epitope interactions Peptide chip analysis was performed to investigate whether the IPA elution buffer had a measurable effect on the epitope– paratope binding. Twenty-five TRIM21 peptides from the central part of the protein sequence (aa161-319 in rhTRIM21) were deposited on glass slides as 15-mers with a frame shift of six amino acids. Staining of the chip with the anti-TRIM21 antibody resulted in a signal of 57 140 ± 14 038 a.u. (max. possible value: 65 535 a.u.) for peptide aa245-259 (amino acid sequence ELEKDEREQLRILGE) using normal incubation conditions with TBS buffer (positive control). This indicated a strong binding of the antibody to the peptide because the control staining, omitting the primary antibody, only showed background levels of 45 ± 21 a.u. for this peptide. The positive control experiment nicely reproduced our earlier results in which we showed that peptide aa245-259 contained an assembled epitope with an αhelical secondary structure (Al-Majdoub et al., 2013a). Interestingly, when the peptide array was treated with a neutral IPA elution buffer after the immune complex between the anti-TRIM21 primary antibody and peptides had been formed, peptide aa245-259 revealed a strong, saturated strong signal (65 310 ± 45 a.u.; deviations are because of background subtraction). By contrast, addition of the acidic elution buffer glycine/HCl instead of the IPA elution buffer reduced the signal intensity to 25 277 ± 11 860 a.u., showing that under acidic conditions, the antibody was at least partially washed away. Although the release of the antibody under acidic conditions was not complete in this experiment, the results show that the presence of IPA in the “soft” elution buffer did not cause a decrease in the binding signal, that is, did not interfere measureably with antibody–epitope binding.

Mass spectrometric studies on protein G´ structure changes

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conditions for protein-G´-containing solvents with neutral pH were found at source temperatures of 60–70ºC and cone voltage settings of 70–80 V. As expected, when protein G´ was dissolved in aqueous ammonium acetate (pH 6.8), only a few rather broad but intense ion signals with relatively low charge states were observed, which is consistent with a native-like conformation of the protein in this solvent. Our ESI-MS analyses show that protein G´ can tolerate the presence of 5% methanol as only ion signals that are indicative for a native-like folded structure are observed. Interestingly, although at neutral pH and without changing the instrument settings, the charge structure of protein G´ adopts a rather different appearance in an IPA-containing solution. Here, the addition of 10% ethanol is because of solubility issues of IPA. As indicated previously, such a low concentration of ethanol is expected to exert only little effects on the stability of antibody–antigen complexes. Nevertheless, the intense ion signals of protein G´ are quite narrow and shifted toward higher charges, indicating a rather denatured conformation. For comparison, the protein G´ spectrum that is obtained from an acidic solution shows completely desolvated narrow ion signals with high charge states, as expected for denatured proteins. Recording the spectrum from the acetic acid/methanol solvent mixture required changing the instrument settings, that is, source temperature of 30–40ºC and cone voltage settings of 40–50 V for best ion yields. Although we have not analyzed protein structure changes of protein A, we assume an analog behavior with respect to IPA exposure because of structural similarities between protein G´ and protein A, particularly with the resemblances in their hydrophobic cores (Olsson et al., 1987).

DISCUSSION Mass spectrometric proteome research with patient sera has been considered revolutionary for the diagnosis of autoimmune



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To test whether IPA had an effect on the protein G´ structure, we performed nano-ESI-MS analyses using different solvent conditions (Figure 6) because ESI-MS has been used for characterization of protein structures and structure changes since many years (Przybylski and Glocker, 1996). The best desolvation

Figure 6. Nano-ESI mass spectrometry of protein G´ dissolved in different buffers. (A) 50 mM aqueous ammonium acetate : MeOH (95:5 v/v), pH = 6.8. (B) 25 mM IPA in 100 mM aqueous ammonium bicarbonate : EtOH : MeOH (90:5:5 v/v/v), pH 7.5. (C) 2% aqueous acetic acid : MeOH (95:5 v/v), pH = 2.6. In each case, 5 μl was loaded. Selected multiply charged ions are labeled with m/z values, and charge states are indicated.

M. AL-MAJDOUB ET AL. diseases (Plebani et al., 2009). Nevertheless, high levels of autoantibodies in autoimmune patients are usually associated with increased cross reactivity and/or “epitope spreading” (Bellone, 2005). As a matter of fact, employing full-length proteins for autoimmune diagnosis remains problematic (Vojdani, 2008). To overcome these limitations, the application of epitope peptides for autoimmune diagnostics has been suggested (Andresen and Grotzinger, 2009). Because of their high sensitivity and specificity, the application of cyclic citrullinated peptides as commercially available tests for patients with rheumatoid arthritis (Zendman et al., 2006) may currently be the best example to promote this peptide-based diagnostic strategy. Obviously, to develop a reliable clinical assay, each peptide (epitope) that should become important has to be analyzed in detail for its antigenic behavior with antibodies from patients. Because of limitations in the current methodology, reducing the number of components in a test system is preferred. With respect to detailed antibody–epitope interaction studies, a oneto-one scenario is regarded optimal for determining binding kinetics, such as on-rates, off-rates, and dissociation constants. Thus, a fast method to isolate immune complexes from complex mixtures is of great help because once an intact immune complex has been prepared, there are many well-developed methods by which detailed molecular studies can be performed (reviewed in (Ngounou Wetie et al., 2013)), such as surface plasmon resonance (Nelson et al., 1997; Müller et al., 1998; Sonksen et al., 1998) and related spectroscopy methods (Pierce et al., 1999; Velazquez-Campoy et al., 2004), native MS (Tito et al., 2001; Rose et al., 2011; Chen et al., 2013; Debaene et al., 2013; Thompson et al., 2013), and cross linking (Sutherland et al., 2008; Petrotchenko and Borchers, 2010; Schwarz et al., 2013). An example for “native” mass spectrometric analysis of an antibody–antigen epitope peptide complex is shown for illustration (Figure 7). Comparing the ion signals of the antibody with those of the antibody–epitope peptide complex shows a

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Figure 7. Nano-ESI mass spectrometry of the anti-TRIM21 antibody and anti-TRIM21 antibody–epitope peptide complex. (A) anti-TRIM21 antibody in 200 mM aqueous ammonium acetate, pH = 7.1. (B) anti-TRIM21 antibody–epitope peptide complex in 200 mM aqueous ammonium acetate, pH = 7.1. Experimental molecular mass of the antibody: 154 266 Da ± 148. Experimental molecular mass of the immune complex: 162 039 Da ± 170. Selected multiply charged ions are labeled with m/z values, and charge states are indicated.


mass increase of ca 7.7 (±0.2) kDa, matching with a 2:1 complex consisting of the epitope peptides and the antibody. In case one was interested in isolating just an antigen, low-pH denaturation works well as long as the antibody is covalently bound to its substrate. Nevertheless, if one was interested in reusing the immobilized antibody, denaturation of the antibody might adversely affect antigen binding after (incomplete) renaturation. Avoidance of any denaturing step is considered less harmful to the antibody functionality. In the design of this study, we aimed at releasing the intact immune complex from the support material (protein A or protein G´) instead of breaking up the paratope–epitope interaction (Antrobus and Borner, 2011). A good example for the latter has been published with imidazole-containing buffers that were described to effectively dissociate the binding between His-tag peptides and anti-His-tag antibodies already at low (ca 50 mM) imidazole concentrations (Müller et al., 1998). Encouraged by this example, we decided the following: (i) to stick to heteroaromatic compounds for our elution tests and (ii) our test complex should consist of the anti-His-tag antibody and a His-tag-containing peptide. The latter was in our case produced by Lys-C digestion of rhGPI and presented as a component of a peptide mixture. The IPA-containing buffer (50 mM, pH 7.0) was successfully used for elution of the immune complex. Because this His-tag-dependent model immune complex survived the chosen elution conditions intact, we extended our investigations, again successfully, to an immune complex consisting of a polyclonal anti-TRIM21 antibody together with its assembled (conformational) epitope peptide. The epitope peptide was presented to the reversibly immobilized antibody in a peptide mixture derived from rhTRIM21 by Lys-C digestion. We speculate that IPA (predominantly because of its structural similarity to tryptophan) interferes with aromatic amino acid residues of protein G´ and/or protein A that are involved in forming the hydrophobic core of these proteins (Derrick and Wigley, 1993). As a result, this interaction should “soften” the core that consequently should change the overall protein G´ conformation, thereby opening up the interaction surface with the antibody. Charge structure alterations have been found indicative for solution structure changes (Przybylski and Glocker 1996), supporting this assumption. Most antibodies bind to protein A or protein G´ through their Fc domains on the heavy chains or by the constant heavy chain domain CH1, which is located in close proximity to the variable domain (VH) (Bouvet, 1994; Derrick and Wigley, 1994). As immunoglobulins are composed of mainly β-sheet-containing domains that are held together by strong hydrogen bonds, neutral IPA-containing solutions should raise no or only small effects on the antibody structure, hence, standing in contrast to “classic” elution systems. Similarly, no or only little effects are expected on epitope peptides from IPA incubation, at least as long as binding of the epitope to the antibody is maintained by polar interactions. The existence of two polarity-related binding type extremes of epitope–antibody reactivities has been suggested by a recently performed binding study with antibody mixtures from commercially available intravenous immunoglobulin preparations that were tested against more than 75 000 epitope peptides (Lustrek et al., 2013). Whether full-length antigen structures will be affected by IPA incubation such that binding to antibodies will be hampered remains to be shown. Nevertheless, epitope excision experiments (Suckau et al., 1990; Hochleitner et al., 2000) showed that removal of most of the antigen structure—except for the epitope parts—was


J. Mol. Recognit. 2014; 27: 566–574

ISOLATION OF ANTIBODY–EPITOPE COMPLEXES possible without disrupting the immune complex, provided that the antibody structure stayed intact. Supported by the epitope chip analysis results and the observed protein G´ structure changes, we consider the successful elution of the antibody–epitope complexes from protein A and/or protein G´ with low salt IPA buffers under neutral pH conditions a major breakthrough in the preparation of intact immune complexes for further analysis.

Acknowledgements We would like to thank Manuela Ruß, Kristin Flechtner, and Elke Schade for excellent technical assistance. We acknowledge the European Union International Research Staff Exchange Scheme grant “MS-LIFE”, the Yemen Government, and the state of Mecklenburg-Western Pomerania, Germany, for financial support.

REFERENCES

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Hochleitner EO, Borchers C, Parker C, Bienstock RJ, Tomer KB. 2000. Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis. Protein Sci. 9: 487–496. Jacobson DL, Gange SJ, Rose NR, Graham NMH. 1997. Epidemiology and Estimated Population Burden of Selected Autoimmune Diseases in the United States. Clin. Immunol. Immunopathol. 84: 223–243. Jankovicova B, Rosnerova S, Slovakova M, Zverinova Z, Hubalek M, Hernychova L, Rehulka P, Viovy J-L, Bilkova Z. 2008. Epitope mapping of allergen ovalbumin using biofunctionalized magnetic beads packed in microfluidic channels: The first step towards epitopebased vaccines. J. Chromatogr. A 1206: 64–71. Jimenez-Castells C, Defaus S, Moise A, Przbylski M, Andreu D, Gutierrez-Gallego R. 2012. Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin. Anal. Chem. 84: 6515–6520. Lorenz P, Kreutzer M, Zerweck J, Schutkowski M, Thiesen H-J. 2009. Probing the epitope signatures of IgG antibodies in human serum from patients with autoimmune disease. In Methods in Molecular Biology, Reineke U, Schutkowski M (eds). Humana Press: Clifton, N.J.; pp. 247–258. Lu Y, Gaskell SJ, Brookman JL. 1998. Epitope mapping of monoclonal antibodies by mass spectrometry: Identification of protein antigens in complex biological systems. J. Am. Soc. Mass Spectrom. 9: 208–215. Lustrek M, Lorenz P, Kreutzer M, Qian Z, Steinbeck F, Wu D, Born N, Ziems B, Hecker M, Blank M, Shoenfeld Y, Cao Z, Glocker MO, Li Y, Fuellen G, Thiesen H-J. 2013. Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins. PLoS One 8: e78605. Macht M, Fiedler W, Kurzinger K, Przybylski M. 1996. Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T. Biochemistry 35: 15633–15639. Müller KM, Arndt KM, Bauer K, Plückthun A. 1998. Tandem Immobilized Metal-Ion Affinity Chromatography/Immunoaffinity Purification of His-tagged Proteins-Evaluation of Two Anti-His-Tag Monoclonal Antibodies. Anal. Biochem. 259: 54–61. Nelson RW, Krone JR, Jansson O. 1997 Surface plasmon resonance biomolecular interaction analysis mass spectrometry. 1. Chip-based analysis. Anal. Chem. 69: 4363–4368. Ngounou Wetie AG, Sokolowska I, Woods AG, Roy U, Loo JA, Darie CC. 2013. Investigation of stable and transient protein-protein interactions: Past, present, and future. Proteomics 13: 538–557. Olsson A, Eliasson M, Guss B, Nilsson B, Hellman U, Lindberg M, UhléN M. 1987. Structure and evolution of the repetitive gene encoding streptococcal protein G. Eur. J. Biochem. 168: 319–324. Papac DI, Hoyes J, Tomer KB. 1994. Epitope mapping of the gastrinreleasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. Protein Sci. 3: 1485–1492. Petrotchenko EV, Borchers CH. 2010. Crosslinking combined with mass spectrometry for structural proteomics. Mass Spectrom. Rev. 29: 862–876. Pierce MM, Raman CS, Nall BT. 1999. Isothermal titration calorimetry of protein-protein interactions. Methods 19: 213–221. Plebani M, Pittoni M, Celadin M, Bernardi D, Mion MM. 2009. Recent advances in diagnostic technologies for autoimmune diseases. Autoimmun. Rev. 8: 238–243. Popescu C, Zamfir A, Dinca N, Petre B-A, Dragusanu M, Przybylski M. 2008. Molecular Recognition Specificity of Anti-3-Nitrotyrosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical



573

Al-Majdoub M, Koy C, Lorenz P, Thiesen H-J, Glocker MO. 2013a. Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21. J. Mass Spectrom. 48: 651–659. Al-Majdoub M, Opuni KFM, Koy C, Glocker MO. 2013b. Facile Fabrication and Instant Application of Miniaturized Antibody-Decorated Affinity Columns for Higher-Order Structure and Functional Characterization of TRIM21 Epitope Peptides. Anal. Chem. 85: 10479–10487. Andresen H, Grotzinger C. 2009. Deciphering the Antibodyome - Peptide Arrays for Serum Antibody Biomarker Diagnostics. Curr. Proteomics 6: 1–12. Ansong C, Miles SM, Fay PJ. 2006. Epitope mapping factor VIII A2 domain by affinity-directed mass spectrometry: residues 497–510 and 584–593 comprise a discontinuous epitope for the monoclonal antibody R8B12. J. Thromb. Haemost. 4: 842–847. Antrobus R, Borner GHH. 2011. Improved elution conditions for native co-immunoprecipitation. PLoS One 6: e18218. Bellone M. 2005. Autoimmune Disease: Pathogenesis. Wiley: Chichester. Ben-David A, Firer MA. 1996. Immunoaffinity purification of monoclonal antibodies: In search of an elution buffer of general applicability. Biotechnol. Tech. 10: 799–802. Bouvet JP. 1994. Immunoglobulin Fab fragment-binding proteins. Int. J. Immunopharmacol. 16: 419–424. Chen J, Yin S, Wu Y, Ouyang J. 2013. Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody-drug conjugates. Anal. Chem. 85: 1699–1704. Debaene F, Wagner-Rousset E, Colas O, Ayoub D, Corvaia N, Van Dorsselaer A, Beck A, Cianferani S. 2013. Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation. Anal. Chem. 85: 9785–9792. Derrick JP, Wigley DB. 1993. Analysis of Bacterial Immunoglobulin-Binding Proteins by X-Ray Crystallography. Immuno. Methods 2: 9–15. Derrick JP, Wigley D B. 1994. The Third IgG-Binding Domain from Streptococcal Protein G: An Analysis by X-ray Crystallography of the Structure Alone and in a Complex with Fab. J. Mol. Biol. 243: 906–918. El-Kased RF, Koy C, Lorenz P, Montgomery H, Tanaka K, Thiesen H-J, Glocker MO. 2011. A Novel Mass Spectrometric Epitope Mapping Approach without Immobilization of the Antibody. J. Proteomics Bioinform. 4: 001–009. Fornsted N. 1984. Affinity Chromatographic studies on antigen-antibody dissociation. FEBS Lett. 177: 195–199. Goward CR, Murphy JP, Atkinson T, Barstow DA. 1990. Expression and purification of a truncated recombinant streptococcal protein G. Biochem. J. 267: 171–177. Griffiths AE, Wang W, Hagen FK, Fay PJ. 2011. Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction. J. Thromb. Haemost. 9: 1534–1540. Guss B, Eliasson M, Olsson A, Uhlén M, Frej A-K, Jornvall H, Flock J-I, Lindberg M. 1986. Structure of the IgG- binding regions of streptococcal protein G. EMBO J. 5: 1567–1575. Hager-Braun C, Katinger H, Tomer KB. 2006. The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen. J. Immunol. 176: 7471–7481. Hecker M, Lorenz P, Steinbeck F, Hong L, Riemekasten G, Li Y, Zettl UK, Thiesen H-J. 2012. Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis: Special Issue on Autoimmunity in the nervous system. Autoimmun. Rev. 11: 180–190.

M. AL-MAJDOUB ET AL. Methods. In Applications of Mass Spectrometry in Life Safety, Popescu C, Zamfir AD, Dinca N (eds). Springer Netherlands: Netherlands; pp. 55–67. Przybylski M, Glocker MO. 1996. Electrospray Mass Spectrometry of Biomacromolecular Complexes with Noncovalent Interactions—New Analytical Perspectives for Supramolecular Chemistry and Molecular Recognition Processes. Angew. Chem. Int. Ed. Engl. 35: 806–826. Reid JD, Holmes DT, Mason DR, Shah B, Borchers CH. 2010. Towards the Development of an Immuno MALDI (iMALDI) Mass Spectrometry Assay for the Diagnosis of Hypertension. J. Am. Soc. Mass Spectrom. 21: 1680–1686. Rose RJ, Labrijn AF, van den Bremer ET, Loverix S, Lasters I, van Berkel PH, van de Winkel JG, Schuurman J, Parren PW, Heck AJ. 2011. Quantitative analysis of the interaction strength and dynamics of human IgG4 half molecules by native mass spectrometry. Structure 19: 1274–1282. Schwarz R, Tanzler D, Ihling CH, Muller MQ, Kolbel K, Sinz A. 2013. Monitoring conformational changes in peroxisome proliferatoractivated receptor alpha by a genetically encoded photoamino acid, cross-linking, and mass spectrometry. J. Med. Chem. 56: 4252–4263. Scofield RH. 2004. Autoantibodies as predictors of disease. Lancet 363: 1544–1546. Selmi C. 2010. Autoimmunity in 2009. Autoimmun. Rev. 9: 795–800. Sinz A, Bantscheff M, Mikkat S, Ringel B, Drynda S, Kekow J, Thiesen H-J, Glocker MO. 2002. Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 23: 3445–3456. Sobott F, Hernández H, McCammon MG, Tito MA, Robinson CV. 2002. A Tandem Mass Spectrometer for Improved Transmission and Analysis of Large Macromolecular Assemblies. Anal. Chem. 74: 1402–1407. Sonksen CP, Nordhoff E, Jansson O, Malmqvist M, Roepstorff P. 1998. Combining MALDI Mass Spectrometry and Biomolecular Interaction Analysis Using a Biomolecular Interaction Analysis Instrument. Anal. Chem. 70: 2731–2736. Stefanescu R, Born R, Moise A, Ernst B, Przybylski M. 2011. Epitope structure of the carbohydrate recognition domain of asialoglycoprotein receptor to a monoclonal antibody revealed by high-resolution proteolytic excision mass spectrometry. J. Am. Soc. Mass Spectrom. 22: 148–157.

Stefanescu R, Iacob RE, Damoc EN, Marquardt A, Amstalden E, Manea M, Perdivara I, Maftei M, Paraschiv G, Przybylski M. 2007. Mass spectrometric approaches for elucidation of antigen antibody recognition structures in molecular immunology. Eur. J. Mass. Spectrom. 13: 69–75. Suckau D, Kohl J, Karwath G, Schneider K, Casaretto M, Bitter-Suermann D, Przybylski M. 1990. Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. U. S. A. 87: 9848–9852. Sutherland BW, Toews J, Kast J. 2008. Utility of formaldehyde crosslinking and mass spectrometry in the study of protein-protein interactions. J. Mass Spectrom. 43: 699–715. Thompson NJ, Rosati S, Heck AJ. 2013. Performing native mass spectrometry analysis on therapeutic antibodies. Methods 1046–2023. DOI: 10.1016/j.ymeth.2013.05.003 Tito MA, Miller J, Walker N, Griffin KF, Williamson ED, Despeyroux-Hill D, Titball RW, Robinson CV. 2001. Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. Biophys. J. 81: 3503–3509. Tsang VCW, Wilkins PP. 1991. Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity. J. Immunol. Methods 138: 291–299. Velazquez-Campoy A, Leavitt SA, Freire E. 2004. Characterization of protein-protein interactions by isothermal titration calorimetry. Methods Mol. Biol. 261: 35–54. Vojdani A. 2008. Antibodies as predictors of complex autoimmune diseases. Int. J. Immunopathol. Pharmacol. 21: 267–278. Zendman AJW, van Venrooij WJ, Pruijn GJM. 2006. Use and significance of anti-CCP autoantibodies in rheumatoid arthritis. Rheumatology 45: 20–25. Zhao Y, Chait BT. 1994. Protein Epitope Mapping By Mass Spectrometry. Anal. Chem. 66: 3723–3726.

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