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Facile Fabrication and Instant Application of Miniaturized AntibodyDecorated Affinity Columns for Higher-Order Structure and Functional Characterization of TRIM21 Epitope Peptides M. Al-Majdoub,† K. F. M. Opuni,† C. Koy, and M. O. Glocker* Proteome Center Rostock, University Medicine Rostock, Rostock, Germany S Supporting Information *

ABSTRACT: Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an “L−E−Q−L” motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen.

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ization lead to the production of commercial affinity systems embedded in pipet tips.19 In contrast, only a few examples have been reported in which neither the antibody nor the antigen were immobilized for epitope mapping.1,6,9,20 Advantages of this procedure are the avoidance of unwanted chemical modifications of the antibody during immobilization (since poorly controlled) and maintenance of “nondenaturing” conditions during all steps of the experiment. Reversible noncovalent antibody immobilization using protein A- or protein G-coated columns have found a vast number of applications in immunotechnology, mostly for affinity chromatography of antibodies from complex biological samples.21,22 The concept of noncovalent immobilization of an antibody on agarose decorated with a protein G/A mixture for mapping the epitopes on small peptides, such as melittin and glucagon-like peptide-1 7−37 (GLP-1 7−37), respectively, has been reported in a single instance.23 Epitope mapping of autoantigens has become important for designing so-called “next generation chip arrays”.24 Diseasespecific epitope peptides need to be identified and characterized

ass spectrometric epitope mapping approaches have been developed as powerful tools to identify molecular details of antigen−antibody interactions.1,2 Various related approaches used for epitope mapping include HPLC,3 chemical modification4,5 of surface exposed residues on the antigen and differential analysis upon shielding by antibody complexation, analysis of hydrogen/deuterium exchange6 differences on the antigen with and without antibody binding, Western blot analysis with chemically produced antigen fragments7 or with fusion proteins8 that contain partial sequences of the antigen, and affinity chromatography-related approaches.9−11 Mass spectrometric epitope mapping analyses are usually divided into epitope excision2,12 and epitope extraction.1,13 The interaction structure analysis principle has been extended to map antibody paratopes11 and to the analysis of carbohydrate binding sites on lectins,14,15 respectively. Mass spectrometric epitope excision or epitope extraction experiments are mostly performed with chemically immobilized antibodies on a support material, thereby generating an affinity column, and successful applications have been reported, for example, by tresyl-activated sepharose magnetic nitrocellulose beads2 or cyanogen bromide-activated sepharose beads,4,12,16−18 to which the antibody was attached. Advantages of this experimental setting are that the affinity column can be reused after regeneration procedures, although not indefinitely. Miniatur© 2013 American Chemical Society

Received: August 6, 2013 Accepted: October 4, 2013 Published: October 4, 2013 10479

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Hamburg, Germany) where placed inside the 96-deep-well plates. A programmable Rainin E4 XLS 8-channel electronic pipet (Mettler Toledo, Gießen, Germany), equipped with 200 μL PureSpeed Protein Tips containing 5 μL of protein A resin (Mettler Toledo, Gießen, Germany) was placed into the 96deep-well plates (containing 0.5 mL Protein LoBind tubes) following a time program as described below. Preparation of the epitope extraction experiment started with filling all necessary solvents into the 0.5 mL Protein LoBind tubes such that the first row contained 100 μL of equilibration buffer (10 mM sodium dihydrogen phosphate/140 mM sodium chloride, pH 7.4), the second row contained 25 μL of antibody solution (from left to right: mouse monoclonal anti-His-tag antibody, mouse monoclonal anti-RA33 antibody, and 25 μL of 10 mM phosphate buffered saline), the third row contained 25 μL of diluted rhGPI Lys-C digest mix, the fourth row contained 100 μL of wash buffer 1 (10 mM sodium dihydrogen phosphate/140 mM sodium chloride in water, pH 7.4), the fifth row contained 100 μL of wash buffer 2 (140 mM sodium chloride in water), the sixth row contained 100 μL of wash buffer 3 (14 mM sodium chloride in water), the seventh row contained 100 μL of wash buffer 4 (0.3 mM TRIS/HCl, 0.25 mM n-octyl-β-D-glucopyranoside), and the eighth row contained 15 μL of elution buffer (3 mM hydrochloric acid, pH 2.5). After having filled in all solvents, the 96-deep-well plate was centrifuged for 30 s to collect all the solutions at the bottom of the Protein LoBind tubes. Next, pipet holders were placed at the second and the eighth tips to (i) hold the 8channel pipet firmly onto the 96-deep-well plate and (ii) to allow the PureSpeed protein tips to be completely immersed into the solutions during the incubation periods. The pipet was moved from one row to another manually between the incubation steps. The program started by initial equilibration of the PureSpeed protein tips using one cycle of aspiration/dispension of the equilibration buffer (in row 1). The antibodies were then captured on the protein A resin (the blank contained only buffer) using six cycles of aspiration/dispension according to the manufacturer’s recommendations (in row 2). The rhGPI peptide mixtures, adjusted to a molar antigen-to-antibody ratio of 2:1, were subsequently brought into contact with the antibodies for 10 min using six cycles (in row 3). Next, nonspecifically bound peptides were washed away sequentially using three cycles, each with 100 μL of wash buffer 1 (in row 4), 100 μL of wash buffer 2 (in row 5), 100 μL of wash buffer 3 (in row 6), and 100 μL of wash buffer 4 (in row 7). The still bound peptides (epitope peptides) were eluted from the protein A-coated pipet tips together with the antibodies using six cycles of 15 μL of the elution buffer (in row 8). One microliter of each of the eluted solutions was used for mass spectrometric analysis (see below). The above detailed description was used also for epitope mapping of rhTRIM21 (Supporting Information Methods file). Paratope Blocking and Epitope Competition Assays. Paratope blocking and epitope competition assays were performed as described in the Supporting Information Methods file. MALDI TOF-MS and QIT TOF-MS/MS analysis. MALDI TOF-MS and QIT TOF-MS/MS mass-spectrometric analyses were performed as described in the Supporting Information Methods file.7,8,9,24

in detail to compose specialized arrays with peptides/epitopes of importance for a given disease (disease-specific arrays), advancing their acceptance in the clinic for patient screening and diagnostics. Here, we describe the development of the epitope mapping approach with either protein A or protein G resins that are embedded in pipet tips and are used in combination with an electronic programmable multichannel pipet for epitope extraction. The advantage of this system is the simple onestep generation of a miniaturized custom-made affinity column that can be instantly applied for mapping epitopes on large (auto)antigen proteins. We developed the protein A- or protein G-based epitope mapping procedure using a monoclonal antihistidine tag antibody together with the His-tag containing recombinant human glucose-6-phosphate isomerase (rhGPI) autoantigen. The procedure was then tested successfully with a polyclonal anti-TRIM21 (TRIM: tripartite motif) antibody directed toward its epitope on the rhTRIM21 (recombinant human TRIM21 protein) autoantigen.24 Although binding affinities of the studied antibodies to neither their antigens nor epitope peptides with varying sequences are known, the method also enabled systematic studies of epitope structures and related binding properties. In all cases, low picomole and even femtomole quantities of the antibodies were applied.

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MATERIALS AND METHODS Antigen Expression and in-Solution Digestion of Recombinant Antigens. The human TRIM21 and human GPI were expressed as recombinant proteins; rhTRIM21and rhGPI, respectively, and in-solution digestion was performed as described in detail in the methods part of the Supporting Information. Synthetic Epitope Peptides. Three 17-mer peptides with the following partial rhTRIM21 sequences were obtained as lyophylized powders (Peptides and Elephants GmbH, Potsdam, Germany). Peptide “A”, LQELEKDEREQLRILGE (molecular mass: 2098.83 Da); peptide “B”, LQPLEKDEREQLRILGE (molecular mass: 2067.72 Da); and peptide “C”, LQELEKDEPEQLRILGE (molecular mass: 2041.08 Da). All peptides were dissolved in 5 mM phosphate-buffered saline (PBS) buffer (pH 8.3) with a final concentration of 500 μM. Stock solutions were kept at −20 °C. Epitope Determination Using Proteolytic Epitope Extraction from Antigen-Derived Peptide Mixtures with Protein A-/Protein G-Immobilized Antibodies. The epitope extraction procedure is described in detail with peptide mixtures derived from LysC-digested rhGPI as antigen and the mouse monoclonal anti-His-tag antibody (AbD Serotec, Düsseldorf, Germany). The anti-His-tag antibody was dissolved in 10 mM phosphate buffered saline (PBS, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), pH 7.4, with a concentration of 1 mg/mL, and was used as the capturing device. The mouse monoclonal anti-RA33 antibody (Sigma, Munich, Germany), dissolved in 10 mM phosphate buffered saline and 15 mM sodium azide, pH 7.4, with a concentration of 1.5 mg/mL, was used as the negative control, and 10 mM phosphate buffered saline served as the background check. Concentrations of the antibodies (400, 100, and 20 fmol/μL) were prepared by diluting the antibody stock solutions with 10 mM PBS. Each epitope extraction experiment was performed in 96-deep-well plates (Mettler Toledo, Gießen, Germany) using three columns in parallel (one for each antibody and one for the buffer control). Protein LoBind tubes (0.5 mL, Eppendorf, 10480

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Peptide Structure Modeling and Visualization. Structure modeling of the peptides was performed using standard settings (Supporting Information Methods file).25,26 Circular Dichroism Analysis. Circular dichroism analysis of synthetic peptides A, B, and C were performed as described in the Supporting Information Methods file. ESI-MS Analyses and Ion Mobility Mass Spectrometry. Off-line flow injection nanoelectrospray ionization (nano-ESI) mass spectrometry was performed with synthetic peptides A, B, and C. A Synapt G2-S instrument (Waters) was used for measuring the drift times of the synthetic peptides A, B, and C using the off-line sprayer. Enolase tryptic digest was used for collisional cross section calculations (Supporting Information Methods file).27,28

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RESULTS Developing the Epitope Extraction Procedure with Noncovalently Immobilized Antibodies. To develop the epitope extraction procedure with antibodies that were noncovalently immobilized on protein G or protein A, we selected an anti-His-tag antibody plus a His-tag antigen pair, since it provides a well-characterized interaction system. Preloaded pipet tips (200 μL PureSpeed protein tips) containing 5 μL of protein G or protein A resin were applied for immobilization of the antibody because they seemed well suitable for downsizing (see below). As expected, immobilization of mouse monoclonal anti-His-tag antibody (epitopespecific antibody; 10 pmol) as well as of mouse monoclonal anti-RA33 antibody (nonspecific antibody as control; 10 pmol) on protein A was very efficient using the manufacturer’s protocols. RhGPI, encompassing 565 amino acids in length (Supporting Information Figure 1), was selected as the antigen of choice because it contained a His-tag at its C-terminus. Digestion of this protein with Lys-C was possible under denaturing conditions and produced a peptide mixture of great complexity, as determined by MALDI-TOF-MS analysis (data not shown). In order not to abolish antibody−antigen/epitope interactions during the epitope extraction experiment, nondenaturing conditions had to be used. Hence, the rhGPI-derived peptide mixture was redissolved in a 100 mM ammonium bicarbonate solution (pH 8.5) prior to performing the experiments. The full details of the entire epitope extraction procedure encompass, after exposing the antibody to the peptide mix, several washing steps to remove nonepitope peptides. A final elution step under acidic conditions follows to recover the epitope peptide(s) (see details in the Materials and Methods section). Using our protocol, eluted epitope peptides were readily analyzed, for example, by MALDI-TOF mass spectrometry. The mass spectra for rhGPI-derived peptide mixtures, first from denaturing conditions and second from nondenaturing conditions (Figure 1A), were very similar, indicating almost no losses of peptides by rebuffering. All major ion signals in the mass spectrum after resolubilization were assigned to partial sequences of rhGPI, yielding 93% sequence coverage. Minor ion signals belonged to carbamidomethylation products that are observed when a protein is stored in buffers with high urea concentrations for longer periods of time.29 Exposing the rhGPI-derived peptide mixture (20 pmol) to the mouse monoclonal anti-His-tag antibody (specific antibody) resulted in a strong binding of the C-terminal peptide (550QQREARVQLEHHHHHH565). Consequently, the corresponding ion signal at m/z 2079.02 was the dominant ion

Figure 1. MALDI-TOF mass spectra of rhGPI peptides. A: Peptide mixture after in-solution digestion with Lys-C. B: Eluted peptides after epitope extraction using the mouse monoclonal anti-His-tag antibody. The C-terminal His-tag-containing peptide 550QQREARVQLEHHHHHH565 produced an ion signal at m/z 2079.02. C: Eluted peptides from exposure of rhGPI-derived peptides to mouse monoclonal antiRA33 antibody as a nonspecific antibody. D: Elution analysis of blank, that is, without antibody. Ion signals are shown in the mass range from m/z 1000 to m/z 4500. A black dot (●) indicates carbamylation, and an asterisk (*), nonidentified ion signals. 2,5-dihydroxybenzoic acid (DHB) was used as matrix.

signal in the spectrum after washing and elution (Figure 1B). Minor ion signals in this spectrum were all found already in the spectrum from the peptide mixture (cf. Figure 1A). By contrast, the spectrum that was recorded after exposure of the rhGPIderived peptide mixture to a nonspecific antibody (mouse monoclonal anti-RA33 antibody) showed the presence of ion signals with rather low intensities only (Figure 1C). The two largest ion signals could be assigned to peptides belonging to rhGPI that were also seen as minor signals in the extraction experiment with the specific anti-His-tag mouse monoclonal antibody (cf. Figure 1B). Finally, checking the background, that is, peptide ion signals upon elution of the exposed rhGPIderived peptide mixture to the protein A pipet tips alone (blank), showed no ion signals (Figure 1D). The sequence of the eluted C-terminal His-tag peptide at m/z 2079.02 (epitope) was confirmed by MS/MS fragmenta10481

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tion, which resulted in B-type ion series (B9−B15) and Y″-type ion series (Y″10−Y″14) with good intensities (Supporting Information Figure 2). It is important to note that mass spectrometric sequencing of the purified epitope peptide can be directly included in the protocol, which is particularly important when unknown epitopes are isolated. The epitope extraction procedure was downsized stepwise with 5, 1, and 0.5 pmol of the mouse monoclonal anti-His-tag antibody immobilized on the protein A resin. The molar ratios of antibody to the rhGPI-derived peptide mixtures were maintained at 1:2 in all epitope extraction experiments. The MALDI-TOF-MS analyses of the eluted solutions from these experiments clearly showed that the C-terminal His-tag peptide (epitope) could be captured by the mouse monoclonal antiHis-tag antibody and detected by mass spectrometry, even with 500 fmol of antibody used for loading on the protein A resin (Supporting Information Table 1). In all spectra, we observed the presence of background signals with low to moderate intensities (Supporting Information Table 1). They were easily identified as background signals by comparison with the spectra that were obtained after exposure of the rhGPI-derived peptide mixture to a nonspecific antibody or to protein A only (blank). Because proof-of-principle was achieved and down-sizing to femtomole quantities was successful, we went on to test the epitope extraction procedure with a real sample. Application of the Epitope Extraction Procedure. The antigen of choice was rhTRIM21, which consists of 546 amino acids (Supporting Information Figure 3). After Lys-C digestion, again, a complex mixture of peptides was obtained that after resolubilization in nondenaturing buffer (Figure 2A) showed intense ion signals in the mass spectra. The dominating ion signals in the spectrum were assigned to partial sequences of rhTRIM21 and to carbamidomethylation products, yielding a sequence coverage of 98%. Because the matching specific antiTRIM21 polyclonal antibody was from rabbit, we chose protein G for immobilizing the antibody. MALDI-TOF-MS analysis after exposure of the rhTRIM21derived peptide mixture to the rabbit polyclonal anti-TRIM21 antibody (specific antibody) showed in the elution solution a strong ion signal at m/z 3498.85. This mass matched that of the rhTRIM21 partial sequence 233NFLVEEEQRQLQELEKDEREQLRILGEK260. In addition, some additional ion signals, all with lower intensities, were observed, indicating that these were from nonspecifically binding peptides (Figure 2B). Comparison of the above explained spectrum with those from epitope extraction experiments with either a nonspecific antibody (rabbit polyclonal anti-Topoisomerase α II antibody; Figure 2C) or without antibody (Figure 2D) showed that, indeed, except for the assumed epitope peptide ion signal, all ions with lower intensities were observed again. The epitope peptide was sequenced by MS/MS fragmentation directly using the target preparation with the elution solution. Intense B-type ion series (B9, B12, B13, B15−B19, B26− B28) and Y″-type ion series (Y″9-Y″11, Y″19-Y″23) were observed, identifying the epitope peptide without ambiguity (Supporting Information Figure 4). The most intense ion signal at m/z 2129.08 (B17) in the MS/MS spectrum indicates a cleavage after aspartic acid (D), which is consistent with mass spectrometric fragmentation behaviors of peptides. The epitope extraction procedure was then downsized using 1, 0.5, and 0.1 pmol of the anti-TRIM21 rabbit polyclonal antibody, respectively. The molar ratio of antibody to

Figure 2. MALDI-TOF mass spectra of rhTRIM21 peptides. A: Peptide mixture after in-solution digestion with Lys-C. B: Eluted peptides after epitope extraction using the rabbit polyclonal antiTRIM21 antibody. The epitope peptide 233NFLVEEEQRQLQELEKDEREQLRILGEK260 produced an ion signal at m/z 3498.85. C: Eluted peptides from exposure of rhTRIM21-derived peptides to the anti-Topo-α-II rabbit polyclonal antibody as a nonspecific antibody. D: Elution analysis of blank, that is, without antibody. Ion signals are shown in the mass range from m/z 1200 to m/z 3600. A black dot (●) indicates carbamylation, and an asterisk (*), nonidentified ion signals. DHB was used as the matrix.

rhTRIM21-derived peptide mixtures was always set to 1:2. The MALDI-TOF-MS analysis of the eluted solutions from these experiments clearly showed that the epitope peptide (233NFLVEEEQRQLQELEKDEREQLRILGEK260) could be observed at m/z 3498.85 down to 100 fmol of immobilized antibody (Figure 3; Supporting Information Table 2). It should be stated that background ion signals were present also at these low amounts of antibody and antigen peptide mixtures. Epitope Competition and Paratope Blocking Experiments. We synthesized peptides “A”, “B”, and “C” to confirm specific binding of the epitope-containing sequence 233NFLVEEEQRQLQELEKDEREQLRILGEK260. All three peptides were matched to the epitope-containing 17-mer sequence 243LQELEKDEREQLRILGE259. This partial sequence contained two 10482

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Encouraged by this result, we spiked-in peptide A into the rhTRIM21-derived peptide mixture to determine whether both peptides were able to bind to the antibody in a competition assay. MALDI-TOF-MS analysis of an equimolar solution of rhTRIM21-derived peptides after LysC digestion and peptide “A” showed again the presence of all previously detected peptide ion signals in the peptide mixture and, in addition, an ion signal of the synthetic peptide at m/z 2098.83 (Figure 4A).

Figure 3. MALDI-TOF mass spectra of rhTRIM21 peptides upon epitope extraction with rabbit polyclonal anti-TRIM21 antibody. A, 1 pmol; B, 500 fmol; C, 100 fmol of the anti-TRIM21 antibody was immobilized on the protein G resin; D, protein G resin without antibody (blank). The molar ratios of rhTRIM21 peptide mixtures to antibodies were 2:1. DHB was used as matrix.

mutual “LEQL” epitope motifs (underlined), one in a sequential manner at the N-terminus and the other one in an assembled (α-helical) manner along the entire peptide.24 Peptide “A” was composed of the original partial sequence (“A”; “as is”: 243LQELEKDEREQLRILGE259). By contrast, in peptide “B”, the glutamic acid residue (E) at position 245 was substituted with a proline residue (P). This amino acid substitution changed the assumed sequential LQEL motif at the N-terminal border of this peptide to LQPL (“B”; “border”: 243 LQPLEKDEREQLRILGE259). Finally, in peptide “C”, the arginine residue (R) at position 251 was substituted by a proline residue (P) because this amino acid substitution in the center of the peptide was expected to change the conformational structure of the peptide (“C”; “center”: 243LQELEKDEPEQLRILGE259) and should thereby abolish the interaction of this peptide with the antibody as a result of steric hindrance at the paratope. The three synthetic peptides were clearly distinguishable using MALDI-TOF-MS by their different molecular masses (Supporting Information Figure 5A). An equimolar mixture of all three peptides (0.2 pmol/μL) dissolved in PBS resulted after desalting and resolubilization in 60% ACN/0.5% acetic acid in a spectrum with nearly identical ion signal intensities for all three peptides. Exposing all three peptides as a mixture in PBS buffer, that is, in a competitive manner to the rabbit polyclonal anti-TRIM21 antibody, showed by MALDI-TOF-MS analysis of the elution fraction that only peptides “A” and “B” were strongly bound to the antibody (Supporting Information Figure 5B). Both peptides have in common that the assembled epitope motif LEQL is present in an extended α-helical conformation, whereas in peptide “C”, the central proline residue should alter the α-helical conformation drastically. The fact that only peptides “A” and “B” were able to strongly bind to the antibody suggested that the sequential LEQL-epitope motif was of lesser importance as compared with the α-helically assembled LEQLepitope motif.

Figure 4. MALDI-TOF mass spectra of rhTRIM21 peptides and spiked-in peptide “A”. A: Equimolar mixture of peptide “A” (m/z 2098.83) and rhTRIM21-derived peptide mixture. B: Eluted peptides after the competition assay using the anti-TRIM21 rabbit polyclonal antibody. C: Eluted peptides after the paratope blocking assay. DHB was used as matrix.

As expected, after elution of the bound peptides from the protein G immobilized anti-TRIM21 antibody, strong ion signals were seen for both, peptide “A” (at m/z 2098.83) and peptide 233NFLVEEEQRQLQELEKDEREQLRILGEK260 (at m/z 3498.85) that was generated by LysC digestion of rhTRIM21 (Figure 4B). In this experiment, both peptides were bound with indistinguishable affinity, indicating similar physicochemical properties. The situation was different, when peptide “A” was preexposed to the rabbit polyclonal anti-TRIM21 antibody. Subsequent addition of the rhTRIM21-derived peptide mixture 10483

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to the antibody showed, after washing and elution of the bound epitope peptide, only one ion signal at m/z 2098.83 for peptide “A” by MALDI-TOF-MS analysis (Figure 4C). Obviously, peptide “A” had completely blocked the paratope from interacting with the rhTRIM21-derived epitope peptide 233 NFLVEEEQRQLQELEKDEREQLRILGEK260 under the chosen experimental conditions. Conformational Characterization Using Ion Mobility Mass Spectrometry, circular dichroism (CD) Spectroscopy, and Structure Modeling. To proof that the synthetic peptides assumed α-helical secondary structures, circular dichroism measurements were performed. These analyses showed that all three synthetic peptides (“A”, “B”, and “C”) were, indeed, mostly α-helical in the tested PBS buffer (Supporting Information Table 3). The three CD curves contained the absorbance maxima at 192 nm and the minima at 210 and 223 nm, respectively. Interestingly, the introduction of proline residues at two different positions did not alter the overall α-helical secondary structure of the peptides, although it is known that proline behaves as an α-helix breaker. To shed more light on this issue, we conducted a structure modeling experiment for the three synthetic peptides using online available sources (see Supporting Information Methods section). The modeled structures are in agreement with the circular dichroism measurements, indicating that peptide “A” assumed a typical α-helix in which the LEQL amino acid residues from the assembled epitope motif are aligned on one and the same side of the α-helix (Figure 5). In general, an extended helical structure is also seen in peptide B; however, with slight bending of the N- and C-termini. Introducing a proline residue at the N-terminus obviously had little effect on the secondary structure. Interestingly, according to the modeling result, peptide “C” assumed an overall α-helical structure, as well, yet that structure consisted of two partial helices located on either side of the central proline residue, forming a U-shaped molecule. Because these calculations indicated that two distinct conformations exist, extended “stick-like” α-helical structures for peptides “A” and “B” and a “U-shape” structure for peptide “C”, we investigated the ion mobilities of the doubly and triply charged peptide ions by nanoESI-IMS-MS. Dissolving the peptides in acetic acid- and acetonitrile-containing solvents provided easily sprayable mild acidic solutions (pH 3.5) and high ionization yields for all three peptides. Peptides “A” and “B” showed doubly, triply, and quadruply charged ion signals with high intensities. For peptide “C”, only the doubly and triply charged ion signals were found with high abundance. In all cases, the triply charged ion was the most intense. Drift time ion mobility mass spectrometry analysis of the triply charged ions showed that peptides “A” and “B” migrated with comparable speeds (∼880 μs), whereas peptide “C” drifted significantly faster (790 μs) under the chosen experimental conditions (Figure 5D; Table 1). The calculated collisional cross section for the triply protonated ion of peptide “A” was 405 Å2; for peptide B, 409 Å2; and for peptide “C”, 370 Å2. This result stands in agreement with the modeling results. The proline residue in the center of peptide “C” obviously caused a smaller overall shape (U-shape), and peptides “A” and “B”, because of their “linear stick-like” conformations, were suspected to appear larger. Repeating the ion mobility experiment under neutral pH solution conditions provided the overall same result, except that the doubly charged ion signals were the dominating ion signals

Figure 5. Nano-ESI mass spectrometry of synthetic peptides “A”, “B”, and “C”. A: peptide “A” (LQELEKDEREQLRILGE; m/z 2098.83). B: Peptide “B” (LQPLEKDEREQLRILGE; m/z 2067.72). C: Peptide “C” (LQELEKDEPEQLRILGE; m/z 2041.08). Multiply charged ions are labeled with m/z values. 3D models of the synthetic peptides indicate extended α-helices for peptides “A” and “B” in which the LEQL motifs are arranged on one side. The peptide “C” model adopts a U-shaped conformation with α-helices on either side of the proline residue. D: Ion-mobility drift time analysis of the three peptides (“A”, “B”, and “C”).

in the mass spectra (data not shown). Interestingly, triply charged ions were observed only for peptides “A” and “B” under these conditions, which is consistent with large enough surfaces to tolerate significant Coulombic repulsion when a third proton is added. Drift times of the doubly charged peptides “A” and “B” (∼1450 μs) were slower than that of peptide “C” (1390 μs). The calculated collisional cross section of the doubly charged ion of peptide “A” was 394 Å2; of peptide “B”, 396 Å2; and of peptide “C”, 338 Å2. In sum, although the α-helical secondary structures seemed well developed in all three peptides, making it difficult to observe differences by circular dichroism measurements, the molecular modeling results matched with both the observed ion mobility drift time/collisional cross section differences and the different antibody binding properties of the investigated peptides. In the end, the existence of an extended α-helix through which the epitope motif L−E−Q−L is assembled on one and the same side of the extended helix seemed the best explanation for all experimental data.

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DISCUSSION Autoimmune diseases, although rare,30 affect young and middle-aged adults with sometimes devastating outcome.31 Autoantibodies targeted against “self antigens” and, hence, 10484

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Table 1. Isoelectric Points of Synthetic Peptides, Drift Times, And Collisional Cross Sections of Multiply Charged Ions at Mild Acidic and Neutral pHa collisional cross section (Å2)

drift time (ms) pH 3.5

a

pH 3.5

pH 6.9

peptide

sequence

[M + 3H]3+

[M + 2H]2+

[M + 3H]3+

pH 6.9 [M + 2H]2+

[M + 3H]3+

[M + 2H]2+

pI

“A” “B” “C”

LQELEKDEREQLRILGE LQPLEKDEREQLRILGE LQELEKDEPEQLRILGE

0.89 0.87 0.79

1.45 1.45 1.39

0.88 0.88 n.d.

1.45 1.45 1.39

405 409 370

394 396 338

4.31 4.41 3.93

n.d., not detected.

are prerequisites for analyses demanding low sample amounts for both, the (typically precious) antibody and the antigen. The combination of an effective affinity chromatography system with mass spectrometry guarantees readout with highthroughput capability, not only coping with the parallel assay format of the affinity system but also providing molecular structure detail information at the same time. Precise and accurate mass determination is feasible with subfemtomol quantities by both MALDI-MS and ESI-MS methods.41,42 MS/ MS fragmentation capability has become routine with modern instrumentation, making mass spectrometry ideal to identify partial sequences and primary structure details of even unknown epitopes. With ESI-MS, particularly in combination with ion mobility analysis, higher-order structures of epitope peptides can be analyzed when these result in drift-time differences,43,44 as shown here with the differentiation of extended α-helical structures from (smaller) U-shaped conformations. In fact, the “charge structure”45,46 of a peptide may be indicative for the respective molecule dimension differences in the gas phase. Although peptide “C” has a calculated pI of 3.93 (Table 1), which is about a half pI unit below that of peptides “A” and “B” (pI ∼ 4.4), it may be speculated that the absence of the quadruply charged ion of peptide “C” in the electrospray mass spectra (cf. Figure 5C) stands in agreement with an overall smaller molecular shape as compared with the more extended “stick-like” shapes of peptides “A” and “B”. However, since the peptides were sprayed from acidic solutions, one must still consider that they resembled denatured random-coil structures of different sizes despite consisting of 17 amino acid residues in each case. Yet, spraying the peptides from neutral pH solutions not only shifted all “charge states” toward lesser protonated ion signals but also showed that the doubly protonated peptides “A” and “B” migrated more slowly than the doubly protonated peptide “C”, again in agreement with the overall shape assumptions of the α-helical structures as obtained by the modeling experiments. The existence of α-helical structures in the gas phase has been reported.44 Notably, our comparison of the here described antigenic behavior of peptide “A” in solution with that when immobilized on the glass surface of peptide chips24 leads to the conclusion that an α-helical secondary structure was presented in either case. Whether epitope chips will assist or even replace antigenbased diagnostic assays in the future relies on the experiences that shall be made with respect to the specificity and sensitivity of these assays. The ultimate performance of a peptide chip in this respect certainly relies on the careful selection of epitope peptides, catapulting reliable and miniaturized epitope mapping procedures into the center of interest.

complete tissues and organs cause severe and chronic morbidity (e.g., rheumatoid arthritis, systemic lupus erythematosus, and rheumatic fever) as well as increased mortality (scleroderma and myocarditis, respectively).32,33 Autoimmune diseases constitute a major challenge and difficulty in clinical diagnosis because the presence of autoantibodies occurs in healthy individuals, as well, and autoantibodies against the same autoantigen can be found in patients suffering from different autoimmune diseases.32−35 Still, current clinical diagnosis of autoimmune diseases relies on cell-based methods, such as indirect immunofluoresence or on (auto)antigen-based methods such as ELISA36 with sometimes unclear results, for example, as a result of unknown promiscuities in the patient’s antibody−antigen interactions. To gain a higher resolution in antibody−antigen related diagnosis, the detailed study of epitope−paratope interactions has been suggested to be implemented into clinical diagnostics.37 Consequently, peptide arrays have been suggested as diagnostic tools in autoimmune diseases.38 Currently, peptide−chip microarrays make use of synthetic peptides (typically 15-mers) with overlapping sequences (e.g., with a frame shift of ∼6 amino acid residues each), covering the full lengths of autoantigen proteins. All peptides are attached to a chip surface (e.g., a glass slide), onto which application of a patient’s serum follows.38,39 However, so-called “next generation chip arrays” should be composed of peptides/epitopes that are known to be of importance for a given disease (diseasespecific arrays), advancing their acceptance in the clinic for patient screening and diagnostics. Such disease-specific epitope peptides, however, need to be identified and characterized in detail, which can only be achieved by highly efficient epitope mapping techniques. The unique properties of the protein G- or protein A-based miniaturized antibody affinity columns in combination with programmable multichannel pipettes, as shown here, provide the flexibility that is needed for such research projects. Bidirectional flow of the sample over the resin bed maximizes the capture of the target antibody and the antigen (mixture) by allowing the binding reaction to reach equilibrium. Using this system, taylored antibody columns were generated in just one step within minutes. These were then instantly ready to use for epitope extraction experiments using a multistep pipetting protocol. Multiple samples (up to eight with our equipment) can be processed in parallel, which provides the advantage that necessary controls and blanks can be analyzed in parallel. Because the binding sites of protein G or protein A on antibodies are known to be located on the constant parts of the antibody,40 the paratope regions, as parts of the variable regions of the immobilized antibodies on the affinity column, remain freely accessible for antigen/epitope binding. Such properties 10485

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ASSOCIATED CONTENT

* Supporting Information

ACKNOWLEDGMENTS

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REFERENCES

Dr. Reiner Würdinger, Dr. Wolfgang Baumann, and Ms. Anja König are acknowledged for their help with CD spectroscopy measurements. Dr. Peter Lorenz and Ms. Nadine Born have helped to express and isolate the recombinant human TRIM21 protein. Dr. Claudia Röwer, Dr. Stefan. Mikkat, and Mrs. Manuela Ruß provided their expertise in mass spectrometry. We thank Dr. Alex Muck for help with the ion mobility MS measurements and Mr. Michael Kreutzer is acknowledged for his assistance with the modeling software. We also acknowledge the Yemen Government and the state of Mecklenburg Western-Pomerania for financial support.

S

Supporting Materials and Methods, five figures and three tables. (SI file 2) Materials and Methods. (SI Figure 1; SI file 6) RhGPI amino acid sequence. The human protein sequence is printed in bold letters (single letter code), and the flanking sequence at the C-terminus, in italics. The black box indicates the sequence that includes the epitope as determined by epitope extraction with the mouse monoclonal anti-His-tag antibody. The underlined partial sequences mark peptides that were nonspecifically bound to either protein A (blank) or to the mouse monoclonal anti-RA33 antibody (nonspecific antibody). (SI Figure 2; SI file 7) MALDI-QIT-TOF-MS/MS analysis after fragmentation of the precursor ion at m/z 2079.02 (cf. Figure 1B). B-type ions and Y″-type ions are labeled. The determined partial sequence matches the C-terminal peptide 550−565 of rhGPI that carries the His-tag. (*) Nonidentified ion signal, (Λ) loss of ammonia, and (#) loss of water. DHB was used as matrix. (SI Figure 3; SI file 8) RhTRIM21 amino acid sequence. The human protein sequence is printed in bold letters (single letter code), and the flanking sequences at the Nand C-termini, in italics. The black box indicates the sequence that includes the epitope as determined by epitope extraction with the rabbit polyclonal anti-TRIM21 antibody. The underlined partial sequences mark peptides that were nonspecifically bound to either protein G (blank) or to rabbit polyclonal anti-Topo-α-II antibody (nonspecific antibody). (SI Figure 4; SI file 9) MALDI-QIT-TOF-MS/MS analysis after fragmentation of the precursor ion at m/z 3498.85 (compare to Figure 3B). B-type ions and Y″-type ions are labeled. The determined partial sequence matches the peptide 233−260 of rhTRIM21. (*) Nonidentified ion signal, (Λ) loss of ammonia, and (#) loss of water. DHB was used as matrix. (SI Figure 5; SI file 10) MALDI-TOF mass spectra of synthetic peptides. A: Equimolar mixture of peptides “A” (LQELEKDEREQLRILGE; m/z 2098.83), “B” (LQPLEKDEREQLRILGE; m/z 2067.72), and “C” (LQELEKDEPEQLRILGE; m/z 2041.08). B: Extracted epitope peptides upon competition between synthetic peptides for rabbit polyclonal anti-TRIM21 antibody binding. DHB was used as matrix. (SI Table 1; SI file 3) His-tag peptide extraction from rhGPI using a protein A-immobilized anti-His-tag monoclonal antibody. (SI Table 2; SI file 4) RhTRIM21 epitope peptide extraction using a protein Gcoupled anti-TRIM21 polyclonal antibody. (SI Table 3; SI file 5) Circular dichroism spectroscopy data of synthetic epitope peptides. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Proteome Center Rostock, University of Rostock, Medical Center and Natural Science Faculty, Schillingallee 69, P.O. Box 100 888, 18055 Rostock, Germany. Phone: +49 - 381 - 494 4930. Fax: +49 - 381 - 494 4932. E-mail: michael. [email protected]. URL: www.pzr.med.uni-rostock. de. Author Contributions †

M. Al-Majdoub and K. F. M. Opuni contributed equally to the manuscript. Notes

The authors declare no competing financial interest. 10486

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