Design of Recombinant Antibody Microarrays for Cell Surface

Nov 30, 2007 - BMC D13, Lund University, SE-221 84 Lund, Sweden, and Bioinvent International AB,. SE-223 70 Lund, Sweden. Received May 4, 2007...
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Design of Recombinant Antibody Microarrays for Cell Surface Membrane Proteomics Linda Dexlin,†,‡ Johan Ingvarsson,†,‡ Björn Frendéus,§ Carl A. K. Borrebaeck,†,‡ and Christer Wingren*,†,‡ Deptartment of Immunotechnology, BMC D13, Lund University, SE-221 84 Lund, Sweden, CREATE Health, BMC D13, Lund University, SE-221 84 Lund, Sweden, and Bioinvent International AB, SE-223 70 Lund, Sweden Received May 4, 2007

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored. Keywords: Antibody microarrays • glycomics • membrane proteomics • membrane proteins • protein microarrays • recombinant antibodies • scFv

Introduction Antibody-based microarrays are among the novel classes of rapidly emerging proteomic technologies, enabling multiplexed protein expression profiling of clinical samples in a highthroughput miniaturized format.1–8 In analogy to gene expression analysis based on DNA microarrays,9,10 global proteome profiling will play a key role in identifying, characterizing, and screening all proteins encoded by the genome.6,7,11 Sequencing of the human genome has revealed about 21 774 genes (http:// www.ensembl.org/Homo_sapiens/index.html), believed to encode approximately 100 000–1 000 000 proteins, defined as the proteome.7,12 To generate complete maps of the entire proteome, both membrane proteins, making up about 30% of the total protein content,13,14 and water-soluble proteins must be analyzed. To date, however, the former group has been difficult to address due to limitations in proteomic technologies.15–18 In this context, it should be noted that membrane proteins and, in particular, the plasma membrane proteome constitute a key group of proteins, and it is one of the most common targets for therapeutics and disease diagnostics.19,20 In addition, mapping of the changes that occur in the cell surface membrane proteome during differentiation, in response to various stimuli, will provide an increased understanding of fundamental processes in both health and disease. While traditional proteomic technologies, including liquid chromatography or two-dimensional gel electrophoresis coupled * Corresponding author: Christer Wingren, Dept. of Immunotechnology, Lund University, BMC D 13, SE-221 84 Lund, Sweden. Phone: +46-46-222 4323. E-mail: [email protected]. † Deptartment of Immunotechnology, BMC D13, Lund University. ‡ CREATE Health, BMC D13, Lund University. § Bioinvent International AB. 10.1021/pr070257x CCC: $40.75

 2008 American Chemical Society

with mass spectrometry,6,7,15,21,22 have made great progress in targeting water-soluble proteins, membrane proteins are still underrepresented in current databases. Alternative techniques, such as ELISA and FACS, are used for plasma membrane analysis, but only for low-throughput approaches. Thus, the need for novel means allowing us to perform high-throughput cell surface membrane proteomics in a facile manner is tremendous. Recently, the first protein23 and antibody24–29 microarray setups for cell surface membrane protein profiling have been presented. The potential of the antibody-based designs was demonstrated by phenotyping leukemic cells24–26 and stem cells,27,28 as well as blood cells.29 Despite their success, the issue of scaling up such arrays will be difficult as the efforts of generating thousands of polyclonal and monoclonal antibodies will be logistically overwhelming.1,2,4,30,31 In comparison, we have during recent years designed a high-performing recombinant antibody microarray technology platform against watersoluble protein analytes.32–39 By using human recombinant single-chain Fv (scFv) antibodies, microarray adopted by design, selected from a large (3 × 1010 members) phage display library,40 we have access to an almost limitless supply of high-performing34,37,38 probes harboring any desired specificities.40,41 In this study, we have designed the first generation of a scaleable human recombinant scFv antibody microarray platform for expression profiling of cell surface membrane proteins as well as cell surface associated carbohydrates on intact cells. To generate a proof of principle, microarrays based on a set of recombinant scFv antibodies were fabricated and used to profile pure or mixed cell populations, including B cells, T cells, dendritic cells, and monocytes. The multiplexed setup was found to display a high specificity and sensitivity, and the Journal of Proteome Research 2008, 7, 319–327 319 Published on Web 11/30/2007

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Figure 1. Proof of principle for the recombinant antibody microarray setup. (A) Structural homology model of the four extracellular domains (D1-red, D2-cyan, D3-pink, and D4-yellow) of wt-CD40 transmembrane protein and two modified CD40 constructs thereof, D1-CD40 and D3-CD40, onto which the specificity of the scFvs has been mapped.42,43 (B) CD40 expression profiles (microarray images) of the three DRAQ5 stained WEHI cell lines expressing wt-, D1-, and D3-CD40, respectively, using a 10 recombinant scFv antibody microarray, 9 scFv specific for the CD40 membrane protein and 1 nonspecific scFv (negative control). (C) Signal intensities corresponding to the array data shown in panel B; gray bars, WEHI wt-CD40; striped bars, WEHI D1-CD40; and black bars, WEHI D3-CD40. The mean signal intensities, based on eight replicates, are shown after subtracting local background. No signals from WEHI D3-CD40 cells could be detected.

results showed that (differential) cell surface membrane proteomics (and glycomics) of intact cells could readily be performed.

Material and Methods Antibodies. Twenty human recombinant scFv antibodies directed against various cell surface membrane proteins, including CD40 (clones 13, 24, 27, 30, 33, 43, 44, 49, and 54),42,43 CD54 (clone CB27), CD154 (clone CB3), HLA-DR/DP (clone CB26), IgM (clones CB28, C10, and B10), and an unidentified membrane protein (sAgX) (clone F1), as well as against cell surface carbohydrates, including Lewisx (Lex) (clones CB15 and CB16), Lewisy (Ley) (clone CB17), and sialic acid modified Lewisx (SiaLex) (clone CB18), were stringently selected from the n-CoDeR library40 and kindly provided by BioInvent International AB (Lund, Sweden). If possible, several validated clones per antigen were applied, to further corroborate the observed reactivity patterns. In addition, three scFv antibodies (clones CT17, TNF-β-19, and IL-8-39) directed against choleratoxin subunit B (CT) , TNF-β, and IL-8 were included and used as negative controls. While 15 of 21 antibodies carried C-terminal FLAG- and his6-tags, 6 of 21 only carried a C-terminal his6-tag (clones CB26, CB27, CB28, C10, B10, and F1). The two different tag designs merely reflected the fact that the antibodies were selected from different generations of the library in which the tag composition had been modified. R-Phycoerythrin (R-PE)-conjugated mouse antihuman CD14 monoclonal antibody (mAb) and biotin-conjugated rabbit antimouse Ig antibody were purchased from DakoCytomation (Glostrup, Denmark), and PE-conjugated mouse antihuman CD22 mAb was from Diatec (Oslo, Norway). Mouse fluorescein isothiocyanate (FITC)-conjugated anti-FLAG mAb and mouse PE-conjugated anti-His mAb were obtained from Sigma-Aldrich (St. Louis, MO) and R&D Systems (Minneapolis, MN), respec320

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tively. Purified mouse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Production and Purification of scFvs. Briefly, all scFvs were produced in Escherichia coli. Soluble scFvs were purified from expression supernatants by affinity chromatography on Ni2+NTA (Qiagen, Hilden, Germany). Bound molecules were eluted with 250 mM imidazole, dialyzed against PBS, and then stored at 4 °C until further use. The integrity and degree of purity of the produced scFvs were evaluated by 12% SDS-PAGE (Invitrogen, Carlsbad, CA). The protein concentrations were determined by measuring the absorbance at 280 nm. Structural Homology Model. A structural homology model of human CD40 was generated using the structure of human TNF receptor (PDB code 1TNR)44 as template. A 13 amino acid long peptide was then modeled onto the N-terminal of the D1 domain using the MacPymol program (http://pymol. sourceforge.net). Cell Cultures. WEHI-231 cells expressing wild-type wt-CD40 and modified constructs thereof (D1-CD40 and D3-CD40)42,43 (Figure 1A) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 2 mM L-glutamine (Invitrogen), and 1 mM 2-mercaptoethanol (Merck, Whitehouse, NJ). The human Burkitt’s lymphoma B-cell line, Ramos (ATCC, CRL-1596), and the human T lymphoblast cell line, Molt-4 (ATCC, CRL-1582), were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM L-glutamine, and referred to as R10 complete medium. The target cells included in the study were selected based on their known differential expression of the target analytes, and their well-known changes in surface expression of these analytes in response to external stimuli. Generation of Monocyte-Derived Dendritic Cells (MoDC). Human peripheral blood mononuclear cells (PBMCs) were purified from leukocyte enriched buffycoats (Lund Uni-

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Membrane Protein Profiling Using Antibody Microarrays versity Hospital, Lund, Sweden), using Ficoll-Paque PLUS density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). CD14+ monocytes were purified by magnetic cell sorting using microbead-conjugated anti-CD14 antibodies (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic cell separation columns (Miltenyi Biotec). The cells were >97% CD14+ as determined by fluorescent activated cell sorting (FACS) analysis. To generate immature MoDCs cells (iMoDC), the purified CD14+ cells were cultured (5 × 105 cells/ mL) for 7 days in R10 complete medium supplemented with 300 ng/mL recombinant human granulocyte macrophagecolony stimulating factor (rhGM-CSF) (Leukine) (Immunex, Seattle, WA) and 100 ng/mL recombinant human IL-4 (rhIL-4) (R&D Systems), and referred to below as monocyte-conditioned medium.45 Every 2–3 days, half of the monocyte -conditioned medium was exchanged. To generate mature MoDCs (mMoDCs), the iMoDCs were stimulated after 7 days of culture for an additional 48 h with 1 µg/mL lipopolysaccharide (LPS) (SigmaAldrich) added to the monocyte-conditioned medium. The DC phenotype was confirmed by monitoring the phenotypic cell surface markers CD1a and CD86, using FACS analysis. FACS Analysis. Suspensions of 2 × 105 cells per sample, suspended in 1% (w/v) BSA in PBS (PBS-BSA), were incubated for 15 min at 4 °C with 15 µg/mL of the different scFvs (specific and nonspecific). PBS-BSA was used in all cell staining and washing steps. After washing, the cells were stained with FITCconjugated anti-FLAG antibody (11 µg/mL) or PE-conjugated anti-His antibody (1.25 µg/mL) in PBS-BSA for 15 min at 4 °C, and then washed. To determine the level of background staining, the anti-FLAG and anti-His antibodies were incubated directly with unlabeled cells. The cell samples were analyzed on a FACScan (Becton Dickinson, San Jose, CA), and data was processed using FlowJo software (Star, Inc., San Carlos, CA). Fabrication of scFv Microarrays. Unless otherwise stated, the antibody microarrays were fabricated by dispensing Ni2+NTA purified scFvs (70-300 µg/mL) onto black polymer MaxiSorb slides (NUNC A/S, Roskilde, Denmark) using the noncontact printer Biochip Arrayer135 (Perkin-Elmer Life & Analytical Sciences, Wellesley, MA). The scFvs were arrayed, in eight replicates each, by spotting 3 drops (333 pL/drop) on top of each other (the spots were allowed to dry out completely before adding the next drop), and individual subarrays were created using a hydrophobic marker pen (DakoCytomation). Subsequently, the subarrays were blocked with 2% (v/v) Tween 20 in PBS (PBS-T) for 2 h and then washed 4 × 2 times with PBS. The slides were immediately used for analysis. In one experiment, anti-CD40 scFv clone 30 was dispensed by hand (1 µL) onto a protein binding glass slide (NUNC), after which the slide was handled as described above. Finally, antibody microarrays were also fabricated on ZeptoMARK Planar Wave Guide (PWG) chips (Zeptosens, Witterswil, Switzerland),46 as described above, with the following exceptions. A chip fluidic structure (containing six fluidic cells, chamber volume of 15 µL) was used to create subarrays. Further, the slides were immediately blocked for 30 min using ZeptoMARK Blocking Buffer BB1 (Zeptosens) and ZeptoMARK Blocking Station (Zeptosens). Next, the slides were rinsed with water and dried under a stream of nitrogen, and subsequently stored at 4 °C overnight prior to use. ScFv Microarray Analysis. Microarray analysis was performed with labeled cells or unlabeled cells. First, 1 × 106 cells were labeled for 10 min at 4 °C in 70 µL of 1 µM DRAQ5 (Biostatus, Leicestershire, U.K.), a permeating fluorescent dye

staining the nuclear DNA of live mammalian cells. Next, the labeled cells were washed two times in 1 mM EDTA in PBS (PBS-EDTA) to remove unreacted dye. The cell samples were then suspended in PBS-EDTA to a density of 3 × 107 cells/mL, before 25 µL was added to each subarray and incubated for 1 h. All incubations were conducted in a humidity chamber at room temperature. Next, the arrays were washed 4 × 2 times with PBS to remove unbound cells. DRAQ5-stained cells bound to the arrayed scFvs were fixated with 10% (w/v) paraformaldehyde in PBS for 30 min and subsequently washed 4 × 2 times with PBS. In the case of unlabeled cells, cell samples were suspended in PBS-EDTA to a density of 3 × 107 cells/mL. Twenty-five microliters of the cell suspensions was then added to each subarray and incubated for 1 h. To perform analysis of mixtures of different cell types, cell samples were mixed 1:1 before adding to the subarrays. To block any Fc-receptors on the cells, mouse IgG (30 µg/mL) diluted in PBS-BSA was added to each array and incubated for 1 h. After washing the arrays 4 × 2 times with PBS, R-PE-conjugated anti-CD14 antibody specific for monocytes (5 µg/mL) or PE-conjugated anti-CD22 antibody specific for B-cells (5 µg/mL) diluted in PBS-BSA was added to the arrays, incubated for 1 h, and washed 4 × 2 times with PBS. Next, the subarrays were fixed with 10% (w/v) paraformaldehyde in PBS as described above. In one experiment, the glass slide arrayed with anti-CD40 clone 30 was incubated 1 h with unlabeled WEHI wt-CD40 cells (3 × 107 cells/mL). After washing the array 4 × 2 times with PBS, biotin-conjugated rabbit antimouse antibody (5 µg/mL) in PBS was added. The arrays were then incubated for 1 h followed by an additional washing step 4 × 2 times with PBS. Finally, the slides were dried under a steam of nitrogen and immediately scanned at 5 µm resolution using the confocal fluorescence scanner, ScanArray Express microarray scanner (Perkin-Elmer Life & Analytical Sciences). The intensity of each spot was quantitated by the fixed circle method using the ScanArray Express software V2.0 (Perkin-Elmer Life & Analytical Sciences). Each data point presented represents the mean value of eight replicates after subtracting local background. In the experiment using the ZeptoMARK protein microarray platform (Zeptosens), 30 µL of DRAQ5-stained WEHI wt-CD40 cells (3 × 107 cells/mL) was added to the printed ZeptoMARK chip via fluidic cells and incubated for 1 h at room temperature. Next, the fluidic cells were washed with 200 µL of assay buffer (0.1% (v/v) Tween 20 and 1% (w/v) BSA in PBS). Subsequently, the microarrays were fluorescence-imaged using the ZeptoREADER instrument, a read-out system based on the PWG technology (Zeptosens).46

Results In this study, we have developed the first generation of a scaleable microarray technology platform, based on recombinant scFv antibody fragments, on black polymer Maxisorb slides for profiling of the cell surface membrane proteome (and glycome). The setup was evaluated to demonstrate the specificity, sensitivity, and applicability of the platform. Proof of Principle. To generate a proof of principle, a 10 recombinant scFv antibody microarray targeting various engineered forms of the cell surface transmembrane protein CD40 was designed and fabricated (Figure 1). Intact WEHI cell lines, expressing either wt-CD40, D1-CD40 (the N-terminal of D1 was extended with a 13 amino acid long peptide), or D3-CD40 (the D1 and D2 domains were deleted) were used as sample. In Journal of Proteome Research • Vol. 7, No. 01, 2008 321

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Figure 3. Proof of principle for an alternative recombinant antibody microarray design. CD40 expression profile (microarray image) of WEHI wt-CD40 cells using the ZeptoMARK protein microarray setup based on planar waveguide detection technology. A five scFv antibody microarray, composed of three scFvs specific for CD40 and two nonspecific scFvs (negative controls; clones TNF-β-19 and IL-8-39) were used. Figure 2. Validation of the recombinant antibody microarray setup. (A) Fluorescence and light microscopy image of wt-CD40 WEHI cells bound to an anti-CD40-specific scFv arrayed onto a transparent glass slide. The cells were stained using biotinconjugated rabbit antimouse Ig and streptavidin Alexa-546. (B) Microarray image (right) and corresponding light microscopy image (left) of DRAQ5-stained MoDc binding to two of five scFv clones arrayed onto nontransparent Maxisorb slide.

Figure 1A, the fine specificity of the 9 anti-CD40 scFv antibodies was mapped onto structural homology models of the three CD40 constructs. The result showed that the microarrays displayed adequate spot morphology (∼150 µm in size) and low background signals (Figure 1B). Further, the specificity profile of the antibodies correlated to 100% with the observed microarray results (cf. panels A and C in Figure 1). In more detail, 9 of 9 antibodies showed binding to wt-CD40+ cells, 4 of 9 bound D1-CD40+ cells, while none of the arrayed antibodies bound D3-CD40+ cells (Figure 1B,C). In all cases, no signals could be observed for the negative control (Figure 1B,C). To further validate the microarray results, the spots were examined in more detail using conventional fluorescence and/ or light microscopy (Figure 2). First, the binding of intact wtCD40+ WEHI cells to an anti-CD40-specific scFv, arrayed onto a transparent glass slide, was examined. The results demonstrated that the fluorescence signal pattern completely overlapped with the visually observed cell binding pattern (Figure 2A). Next, we compared the microarray image obtained for a 5 scFv microarray, fabricated on the nontransparent black polymer Maxisorb slide, targeting CD40+/Lewisx+/sAgX-/IgMmonocyte-derived dendritic (MoDC) cells, with the corresponding light microscopy image (Figure 2B). The results showed that the anticipated scFv antibodies, 2 of 5, bound MoDC cells, and that the microarray image coincided with the light microscopy image. Similar results were obtained whether any of the top four expressing anti-CD40-specific clones (clones 24, 27, 30, and 44) were analyzed (data not shown). Hence, the results showed that cells were specifically bound and correctly detected. To explore whether membrane protein profiling was possible on an alternative platform, intact wt-CD40+ WEHI cells were also analyzed on a 5 recombinant scFv antibody microarray on ZeptoMARK glass slides interfaced with a planar waveguide (PWG) detection technology (Figure 3). Adequate spot morphologies and low to medium background signals were observed (Figure 3). Similar to the data generated on the Maxisorb platform, the results showed that the 3 CD40-specific scFv antibodies gave strong signals, while the 2 negative controls 322

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Figure 4. Validation of the recombinant antibody microarray setup. The antibody microarray-determined cell surface membrane protein (black) and carbohydrate (blue) profiles of Ramos B-cells and monocytes were compared with the corresponding profiles obtained by conventional FACS analysis using the same set of scFv antibodies. A 13 recombinant scFv antibody microarray, composed of 9 membrane-protein-specific clones and 4 carbohydrate-specific clones, was used. Microarray images are shown for DRAQ5-stained Ramos B-cells and CD14+ monocytes. The histograms (signal intensity (x-axis) vs number of cells (yaxis)) represent the FACS staining of each scFv clone (red curve) compared to the negative staining control (blue curve). n.d. ) not determined.

gave no signals (cf. Figures 1 and 3). In conclusion, the data showed proof of principle for recombinant antibody microarrays for plasma membrane protein profiling interfaced with two detection principles. Validation of the Antibody Microarray Platform. To further corroborate the platform, a diverse set of cell surface antigens was selected, including 6 membrane proteins (CD40, CD54, CD154, HLA-DR/DP, IgM, and sAgX) and 3 carbohydrates (Lewisx, Lewisy, and sialic acid modified Lewisx). These were differentially expressed on three different cell types, that is, Ramos B-cell line, CD14+ monocytes, and Molt-4 T-cell line, and were analyzed on a 13 scFv recombinant antibody microarray (Figure 4). In addition, the acquired microarray data were compared with data obtained by conventional FACS analysis using the same set of scFv antibodies. Representative results for two (clones 30 and 44) of the four top expressing anti-CD40-specific scFv clones (clones 24, 27, 30, and 44) are shown.

Membrane Protein Profiling Using Antibody Microarrays

Figure 5. Limit of detection for the recombinant antibody microarrays. Representative results obtained for two cell lineages and three scFvs are shown. (A) Titration curve of the WEHI wtCD40 cells binding to anti-CD40 clones 44 (filled squares) and 30 (open squares). (B) Titration curve of the Ramos B-cells binding to anti-IgM (clone B10). In all cases, the mean signal intensities, based on eight replicates, are shown after subtracting local background.

The microarray results showed that three distinct binding profiles, that is, cell surface antigen expression profiles, matching the expected profiles of the different cell types were obtained (Figure 4). In more detail, the Ramos B cell line bound 11 of 13 arrayed scFv antibodies, demonstrating cell surface expression of CD40, CD54, HLA-DR/DP, IgM, sAgX, Lewisx, and sialic acid modified Lewisx. Further, the CD14+monocytes bound 5 of 13 scFv antibodies, showing that CD54, HLA-DR/ DP, Lewisx, and sialic acid modified Lewisx were expressed on the cell surface. In contrast, the Molt-4 T cell line was only found to express Lewisx (data not shown). When profiling the cells using conventional FACS analysis, an identical expression pattern was observed as in the microarray analysis (Figure 4). Thus, the results confirmed that the platform was capable of targeting both cell surface membrane proteins as well as carbohydrates differentially expressed on different cell types. Limit of Detection. The limit of detection (LOD), in terms of number of cells added to an array, was determined by analyzing serial dilutions of wt-CD40+ WEHI cells (Figure 5A) and IgM+Ramos B-cells (Figure 5B) on a 3 scFv microarray composed of two CD40-specific scFv clones and one IgMspecific clone. The results showed that the LOD was about 12.5 × 104 cells (clone 30) and 1.25 × 104 cells (clone 44) for wtCD40+ WEHI cells (Figure 5A). In comparison, clone 44, that displayed a 10 times lower LOD value, was also found to give a higher positive signal in conventional FACS analysis (cf. Figures 4 and 5). Further, the LOD was found to be around 2.5 × 104 cells, corresponding to approximately 500 cells/spot, for IgM+Ramos B-cells (Figure 5B). Hence, the results implied that the LOD was dependent on the individual scFv clone and/or cell type, that is, expression levels of the target membrane protein. To ensure that we were well above the LOD, 7.5 × 105 cells/array or about 15 700 cells/spot were in general applied throughout the remaining part of the project. Profiling of Mixed Cell Populations. Next, we explored the possibility of profiling also mixed populations of cells by

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Figure 6. Profiling of pure vs mixed cell populations. A microarray composed of 15 scFvs directed against 11 cell surface membrane proteins, 3 carbohydrates, and 1 nonmembrane-protein-specific (negative control) was adapted. (A) Microarray image obtained for pure DRAQ5-stained CD22+ Ramos B-cells. (B) Microarray image obtained for mixed (1:1) populations of CD22+ Ramos B-cells and CD14+ monocytes, visualized using a labeled antiCD22-specific antibody. (C) Microarray image obtained for pure DRAQ5-stained CD14+monocytes. (D) Microarray image obtained for mixed (1:1) populations of CD22+ Ramos B-cells and CD14+ monocytes, visualized using a labeled anti-CD14-specific antibody.

detecting microarray-captured cells with fluorescently labeled phenotype specific antibodies. In Figure 6, mixed populations of CD22+ Ramos B-cells and CD14+ monocytes (1:1) were profiled on a 15 scFv antibody microarray, targeting 6 membrane proteins, 3 carbohydrates, and 1 nonmembrane protein (negative control). The microarrays were visualized using fluorescently labeled anti-CD22 (Figure 6B) or anti-CD14 antibodies (Figure 6D), respectively. The microarray profiles were compared with those obtained for the corresponding populations of pure, nucleus-stained cells (Figure 6A,C). The results showed that identical cell surface membrane protein expression profiles were obtained whether pure or mixed populations of CD40+/CD54+/HLA-DR-DP+/IgM+/ sAgX+ Ramos B-cells (cf. panels A and B in Figure 6) and CD54+/HLA-DR-DP+ monocytes (cf. panels C and D in Figure 6) were analyzed, respectively. Similarly, identical profiles were observed for the cell surface associated carbohydrates, Lewisx, whether mixed or pure cell populations were analyzed, whereas sialic acid modified Lewisx could no longer be detected on either cell type when the complexity of the sample was increased. The negative control was not affected by the multiplexing. Hence, the results showed that cell surface membrane proteomics could be performed on mixed cell populations, simultaneously providing information about both the phenotype and lineage of the microarray captured cells. Profiling of Cell Surface Antigen Expression in Response to Cell Activation. Finally, we examined whether the recombinant scFv antibody microarray platform could be used to monitor the changes in cell surface antigen expression in response to cell activation. To this end, CD14+monocytes were sequentially activated to immature monocyte-derived dendritic cells (iMoDC) and mature monocyte-derived dendritic cells (mMoDC). These three cell types were then analyzed on a 7 scFv antibody microarray, targeting 4 cell surface membrane proteins and one carbohydrate (Figure 7). In addition, the microarray results were compared with data acquired by conventional FACS analysis using the same set of scFv antibodies. Journal of Proteome Research • Vol. 7, No. 01, 2008 323

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Figure 7. Profiling of cell surface antigen expression in response to cell activation. Monocytes (gray bars) were sequentially activated to immature monocyte-derived dendritic cells (iMoDc) (purple bars) and mature monocyte-derived dendritic cells (mMoDC) (red bars), and phenotyped. The antibody microarray-determined cell surface membrane protein and carbohydrate profiles were compared with the corresponding profiles obtained by FACS analysis using the same set of scFv antibodies. A 7 recombinant scFv antibody microarray, composed of 6 membrane-protein-specific clones and 1 carbohydrate-specific clone, was used. The antibody microarray signal intensities (based on eight replicates corrected for local background) and matching microarray images are shown as well as the MFI Geo. Mean signal intensities obtained by FACS staining.

The microarray results showed that the expression levels of CD40 increased in the order of monocytes < iMoDc < mMoDC (Figure 7). The results were consistent for all three CD40specific scFv clones and agreed with the FACS results. Furthermore, the antibody microarray-determined profiles of CD154 (not expressed), CD54 (constant expression), and Lewisx (iMoDC < mMoDC < monocytes) were also found to correlate with the corresponding FACS data. While FACS analysis showed that the expression of HLA-DR/DP increased upon cell activation, the microarray analysis implied a high and constant expression. This discrepancy may, however, be explained by the fact that we were operating at the high end of the dynamic range of the microarray assay, indicating the need of analyzing the sample at serial dilutions for this particular analyte (data not shown). Taken together, the results showed that we have developed a recombinant antibody microarray platform for plasma membrane proteomics (and glycomics) of intact cells.

Discussion Novel technologies for multiplexed, specific, and sensitive profiling of membrane proteins in complex proteomes will play a major role within biomedicine.6,7,20 The first antibody and protein microarray designs targeting membrane proteins and/ or carbohydrates on intact mammalian cells23–29 and bacteria cells47,48 have only recently been published. Initially, these antibody microarray setups, based on antisera or purified intact polyclonal and monoclonal antibodies, have been used to perform mammalian cell phenotyping24–29 or serotyping of bacteria.47,48 Albeit promising, the issue of scaling up such array designs is not trivial, as the efforts of generating thousands of monoclonal and polyclonal antibodies displaying not only the desired specificities but also high on-chip performances will be demanding.1,2,4,30 In this study, we have expanded our current antibody microarray platform targeting water-soluble proteins32–39 and 324

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designed the first generation of a recombinant scFv antibody microarray setup for expression profiling of cell surface membrane proteins as well as cell surface associated carbohydrates on intact cells. Of note, by using an antibody library with 3 × 1010 probes,40,41 microarray-adapted by design to display, for example, high on-chip performances,1,37,38,49 we have thus evolved a readily scaleable platform, in contrast to the conventional antibody microarray setups.24–29,47,48 When scaling up the arrays, the spot size is also a key feature. Compared to the first polyclonal and monoclonal antibody array designs,24–28 our spots are more than 3 times smaller, that is, a diameter of 150 µm versus 500 µm. Assuming a spotto-spot distance of 50 µm, this would theoretically give us about a 140 times higher spot density, 2800 versus 20 spots/cm2. Despite reducing the spot size, the LODs were found to be better, or comparable, to those observed for the previous array designs.24–28 As for example, Belov and co-workers found the LOD to be about 370 cells/mm2,24–26 while we observed LODs in the 230 cells/mm2 range (Figure 5). The improved sensitivity may be explained by the high on-chip functionality of our probes1,37,38,49,50 combined with the fact that the platform was found to display adequate spot morphology, low background, and minimal nonspecific binding. Notably, the assay performance was maintained, or even improved, although we used small (28 kDa, 45 Å long) monovalent scFvs instead of large (150 kDa, 140 Å long) intact bivalent antibodies as probes. Thus, the results implied that both avidity effects and sterical hindrances were of less importance when the arrayed scFvs bound to the multicopy antigens exposed on the cell surface. Previously, the functionality and specificity of polyclonal and monoclonal antibodies in microarray applications has been debated.8,50–52 As for example, Campbel et al. evaluated several intact IgG and IgM antibodies as probes for membrane protein profiling recently and found that the choice of antibody was not

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Membrane Protein Profiling Using Antibody Microarrays obvious, since many antibodies with similar solution reactivity differed in performance on a microarray surface.29 In contrast, we have repeatedly demonstrated the specificity and on-chip functionality of our microarray-adapted scFvs, even when targeting low-abundance protein analytes in directly labeled complex proteomes, such as human serum and plasma.1,32–35,49 In accordance, the stringently selected scFvs used in this study, targeting cell surface membrane proteins and cell surface associated carbohydrates, was also found to display adequate specificity (e.g., Figures 1, 4, and 7). The observed phenotypes also agreed well with the expected expression profiles previously found using conventional methods, such as FACS.45,53 Furthermore, the onchip reactivity correlated very well with the observed solution reactivity of the scFvs, as determined by FACS. From a semiquantitative point of view, the FACS signals and the microarray intensities correlated well when analyzing different antibody clones targeting the same antigen (e.g., the anti-CD40-CD40 system) (cf. Figures 4 and 5) or the same antibody clone targeting differentially expressed antigen on different cell types (Figure 7). The only observed discrepancy, which was noticed when the up-regulated levels of HLA-DRDP were monitored on activated cells (Figure 7), could be explained by the fact that we were operating at the highend of the dynamic range for this particular analyte, indicating a tentative need of analyzing serial dilutions of a sample. However, when looking only at the microarray data, the observed differences in array signal intensities between different antigen–antibody pairs could not be directly transformed into quantitative differences (e.g., Figure 7). This lack of correlation could be explained by differences in (i) the amount of antibody arrayed (see Material and Methods), (ii) on-chip antibody affinities, and (iii) antigen expression levels. Similarly, the same lack of correlation has commonly been observed also for antibody arrays targeting water-soluble proteins.33 In that particular case, differential labeling of the protein analytes was also a major contributing factor. To date, protein microarrays for cell surface membrane proteomics of mammalian cells has been fabricated on polyacrylamide film-coated glass slides,23 activated cellulose membrane,28 patterned monolayer of alkanethiols self-assembled on gold-evaporated glass slides,27 gold-coated glass slides,29 or FAST-slides24–26 and interfaced with optical or fluorescent imaging detection systems.23–29 In the case of fluorescent-based readout, the cells were detected by either (i) direct or indirect labeling of the cells24–28 or (ii) a label-free manner taking advantage of the cells’ autofluorescent properties.29 Here, we expanded the range of competitive fluorescent-based setups by interfacing black polymer Maxisorb slides with a confocal scanner and ZeptoMARK glass slides with a PWG scanner. Of note, the PWG technology has previously been shown to provide excellent sensitivity in the case of antibody/protein microarrays targeting water-soluble protein analytes.46,54 As in all antibody/protein microarray setups, the sample format is a key factor.33,55 An advantage of targeting the cell surface membrane proteome in the format of intact cells, a concept that could also be extended to intracellular organelles, is that the structural integrity and functional properties of the proteins are likely to be maintained. This was, for example, explored by Ko et al. who performed cell proliferation assays on microarray captured cells.27,28 Expanding the range of alternative sample formats, we have recently taken the first steps toward designing a novel setup providing the means of

isolating both the water-soluble as well as the membrane protein subproteomes from a sample under native conditions suitable for microarray profiling (Dexlin and Wingren, unpublished observations). In addition, membrane proteins could also be targeted in reversed affinity arrays, where lysed, denatured cell proteomes have been arrayed46 (Dexlin and Wingren, unpublished observations). Finally, focusing on the glycome instead of the proteome, which has also gained significant biomedical interest entering the postgenomic era,56,57 we demonstrated for the first time that recombinant antibody microarrays potentially could be used for cell surface glycomics. In comparison, intact monoclonal antibodies29 and lectins58,59 are other tentative probes for such applications. Further work will be required to elucidate the impact of recombinant antibody arrays, or arrays based on other carbohydrate binding protein modules, some of which are already available in a library format,60 on the field of glycomics. Taken together, we have designed a semiquantitative technology platform for rapid profiling of the plasma membrane proteome, which in the long run could provide us with unique means to generate detailed maps of the membrane proteome. Although additional technical endeavors will be required to fully explore and exploit issues related to sensitivity, dynamic range, and multiplexity (i.e., targeting of numerous cell types in crude mixtures), the work has clearly outlined the potential of the setup. Further, differential expression profiling of clinical samples from healthy versus unhealthy patients could enable us to rapidly detect multiplexed disease-specific membrane protein signatures that will have an impact in disease proteomics, including, for example, disease diagnostics, biomarker discovery, tumor profiling/ monitoring, and patient stratification. Abbreviations: CT, choleratoxin subunit B; iMoDc, immature monocyte-derived dendritic cells; Lex, Lewisx; Ley, Lewisy; LOD, limit of detection; mAb, monoclonal antibody; mMoDC, mature monocyte-derived dendritic cells; MoDC, monocyte-derived dendritic cells; PE, phycoerythrin; PWG, planar waveguide, sAgX, surface antigen X; scFv, single-chain fragment variable; SiaLex, sialic modified Lewisx.

Acknowledgment. This study was supported by grants from the Gunnar Nilsson’s Cancer foundation, the Swedish National Science Council (VR-NT), the SSF Strategic Center for Translational Cancer Research (CREATE Health), The Swedish Medical Association (the National Board of Health and Welfare), and the Magnus Bergwall foundation. We acknowledge the technical assistance of Marie Jönsson with the fluorescence microscopy analysis. References (1) Wingren, C.; Borrebaeck, C. Antibody microarrays-current status and key technological advances. OMICS 2006, 10 (3), 411–427. (2) Wingren, C.; Borrebaeck, C. A. High-throughput proteomics using antibody microarrays. Expert Rev. Proteomics 2004, 1 (3), 355–364. (3) Borrebaeck, C. A. Antibody microarray-based oncoproteomics. Expert Opin. Biol. Ther. 2006, 6 (8), 833–838. (4) Kingsmore, S. F. Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discovery 2006, 5 (4), 310–320. (5) Haab, B. B. Methods and applications of antibody microarrays in cancer research. Proteomics 2003, 3 (11), 2116–2122. (6) Hanash, S. Disease proteomics. Nature 2003, 422 (6928), 226–232. (7) Zhu, H.; Bilgin, M.; Snyder, M. Proteomics. Annu. Rev. Biochem. 2003, 72, 783–812. (8) MacBeath, G. Nat. Genet. 2002, 32 (Suppl.), 526–532.

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