Aptamer Microarray Mediated Capture and Mass Spectrometry

Aug 31, 2010 - Aptamer Microarray Mediated Capture and Mass Spectrometry Identification of Biomarker in Serum Samples ... S.K.: (phone) +82-2-2260-384...
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Aptamer Microarray Mediated Capture and Mass Spectrometry Identification of Biomarker in Serum Samples Ji-Young Ahn,† Sang Wook Lee,‡ Hye Suk Kang,§ Minjoung Jo,† Dong-ki Lee,*,§ Thomas Laurell,*,†,‡ and Soyoun Kim*,† Department of Biomedical Engineering, Dongguk University, 3-26 Phill-Dong, Joong-Gu, Seoul, 100-715, Korea, Department of Measurement Science and Industrial Electrical Engineering, Divison of Nanobiotechnology, Lund University, Lund, Sweden, and Department of Chemistry, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Korea Received March 31, 2010

Sensitive detection of molecular biomarkers in clinical samples is crucially important in disease diagnostics. This paper reports the developement of an aptamer microarray platform combined with sol-gel technology to identify low-abundance targets in complex serum samples. Because of the nanoporous structure of the sol-gel, a high capacity to immobilize the affinity specific aptamers is accomplished which allows binding and detection of target molecules with high sensitivity. The captured protein is digested in situ and the obtained digest was analyzed by ESI-MS without any interference from the affinity probe. TBP (TATA Box Protein) and its specific aptamers were chosen as a model system. A proof of concept with protein concentrations ranging between nanomolar to micromolar is reported, showing a good linearity up to 400 nM when characterized in an aptamer sandwich assay. Moreover, as low as 0.001% of target protein present in total serum proteins could be identified without any pretreatment step using ESI MS/MS mass spectrometry. We believe this novel strategy could become an efficient method for aptamer-based biomarker detection linked directly to mass spectrometry readout. Keywords: aptamer microarray • sol-gel immobilization • sandwich assay • biomarkers • ESI MS/MS mass spectrometry

Introduction Biomarkers are commonly used as indicators of a biologic state. Especially, secreted disease related compounds and surface exposed proteins from cancer cells can be powerful biomarkers for early detection of the disease as well as cancerstaging without a biopsy treatment.1 Since biomarkers are generally found at low-abundance levels, highly sensitive detection technologies are needed to measure the expression level of a biomarker in complex samples.2 Recently, monoclonal antibody (mAb)-based biomarker discovery platforms have been developed.3 One of the most powerful advantages of mAb is its ability to specifically enrich the target (antigen) in a sample. Using this technology, several groups have tried to do relative or absolute quantification of biomarker candidate proteins by means of mass spectrometry (MS) readout.4-7 However, antibodies might be a severe interference when utilizing MS as the analytical readout for protein identification, since the concentration of captured marker proteins commonly is extremely low in relation to the antibody. In other words, * To whom correspondence should be addressed. T.L.: (e-mail) Thomas. [email protected]. D.-k.L.: (e-mail) [email protected]. S.K.: (phone) +822-2260-3840, (fax) +82-2-2260-3840, (e-mail) [email protected]. † Dongguk University. ‡ Lund University. § Sungkyunkwan University.

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the detection signals of a target protein might be buried under abundant antibody signals. This becomes evident after digesting the antigen/antibody complex including unoccupied antibodies. Therefore, new capturing probes that are not based on a protein scaffold are needed, which do not generate interfering peptide sequences as the target protein/affinity probe complex is digested. In this perspective, aptamers are emerging as novel and highly potent protein binders that fulfill the above criterions. Aptamers are DNA (Deoxyribonucleic acid)/RNA(Ribonucleic acid) oligonecleotides with binding affinity like antibodies, and are stable under a variety of experimental conditions, which enables them to be used as a platform for biosensor development.8 Their tertiary structures are important because they can recognize the 3-dimensional (3D)-structure of target molecules. Moreover, specific aptamers can be readily developed by means of library based selection strategies, for example, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process,9,10 which opens the route to a broadband multiplex approach to aptamer based affinity probing. To capture sufficient amount of low-abundance species in biomarker discovery research, the capture probe not only has to be highly specific and display a high affinity constant, but commonly also a sufficient capture capacity is required for the analytical readout why high surface area matrices such as bead10.1021/pr100300t

 2010 American Chemical Society

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based capturing protocols many times are employed. To avoid the need for micro bead processing, monolithic porous microstructures for protein digestion and analyte enrichment have been frequently reported and used in flow-through configurations.13,14 In the case of affinity specific probing and coupling to mass spectrometry, immuno-MS, the porous matrix not only has to have good capacity in retaining the affinity probe without loosing its affinity, but also at the same time provide unrestricted access for the antigen. Recent work by Kim et al.15 has demonstrated the possibility to incorporate HIV (Human Immunodeficiency Virus)-antigen in sol-gel matrices with maintained target affinity and perform antibody specific binding in serum samples. Sol-gel entrapment of polyclonal antibody (pAb) against HIV-antigen was spotted in a microarray format and the subsequent sandwich assay demonstrated femtomolar detection limit in a fluorescent readout. In this paper, we for the first time present a biomarker discovery platform based on sol-gel incorporated aptamer for the rapid identification of target biomarkers in a complex clinical sample using Liquid Chromatography ElectroSpray Ionization tandem Mass Spectrometry (LC-ESI MS) readout. A three-dimensional nanoporous sol-gel matrix was used in a microarray format since it enables the entrapment of aptamers in their native form, utilizing the high surface area of the sol-gel, for the interaction and thus antigen capturing capacity.15 Starting with serum samples spiked with TATA Binding Protein (TBP), we combined sol-gel microarrays with a highly TBP-specific aptamer as a capturing agent and subsequent on target digestion followed by LC-MS/MS identification of the target protein.

Materials and Methods Protein and Aptamer Preparation. The yeast TATA Box Protein expression system was a generous gift from John T. Lis (Cornell University, Ithaca, NY).16,17 Recombinant fulllength 6× His-tagged yTBP proteins (∼25 kDa) were purified from BL21-DE3 cells using a Ni-charged affinity resin and minicolumn (Qiagen, Hilden, Germany). The purified target protein fractions were dialyzed overnight at 4 °C against 1 L of dialysis buffer (20 mM Tris-HCl, 50 mM KCl, and 10% glycerol, pH 8.0). To confirm the dialyzed protein remained intact, SDS-PAGE analysis was performed. For preparing the aptamers, we used in vitro transcription system according to the manufacturer’s protocol (MEGAshortscript Kit, Ambion). The sequences of the aptamers were as follows: The TBP specific aptamer (#12, 5′GGG AGA AUU CAA CUG CCA UCU AGU GGU AAA CCA CGG GUA ACG GAU AGG AAG UUG UAU UGC CCU AGU ACU ACA AGC UUC UGG ACU CGG U-3′; and #24, 5′-GGG AGA AUU CAA CUG CCA UCU AGG ACA AGG UAA UUA GAC GGC AAG AGA AUA AAC GAG GUC CCA CCA GCA UCG CAG UAC UAC AAG CUU CUG GAC UCG GU-3′) and anti-Hepatitis C Virus (HCV) core specific aptamer, also denoted anti-core aptamer henceforth, (9-15, 5′-GGG CCG TTC GAA CAC GAG CAT GTT GTC TAC GTT GTA GAA GCT GTT ATG GTA GGT ACT TCC ACG AGG TAT CAA CGG AGT TGG TGG ACA GTA CTC AGG TCA TCC TAG G-3′).18 In detail, after amplifying the aptamer DNA construct, each PCR product was purified with QIAquick Purification Kit (Qiagen, Germany), and then, 1 µg of the DNA templates was in vitro transcribed to RNA for 4 h using a MEGAshortscript kit and precipitated directly with 2 vol of ethanol at -20 °C for 2 h. These aptamers were labeled at their 3′-end by terminal deoxynucleotidyl transferase (TdT). In detail,

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1 nmol of RNA aptamer (#24) was incubated for 4 h at 37 °C with 100 µM Cy3-dUTP (E-biogen, Korea), and then 20 units of TdT enzyme (Fermentas) and 10 units RNase inhibitor (Boehringer Mannheim) were added and incubated for 30 min at 37 °C to prepare the final labeled RNA. Microarray Preparation. We utilized the sol-gel materials for immobilizing aptamers according to manufacturer’s recommendation (SolB complete kit, PCL Inc., Korea, www. pclchip.com). In detail, using three sol-gel formulations (F-I: SolB I 2.3%, SolB II 2.3%, SolB III 4.6%, SolB H 18%, SolB S 18%; F-II: SolB I 25%, SolB II 7.5%, SolB III 5%, SolB H 12.5%, SolB S 12.5%; and F-III: SolB I 26.2%, SolB II 10.5%, SolB III 7%, SolB H 12.5%, SolB S 11%,),15 the 200 µM aptamers described above (anti-core aptamer and #12) were mixed with SolB reagents and arrayed with controls (N, negative control; and P, reference control with Cy-3 dUTP) using the non-contact microdispensing instrument (DW-SolB, PCL, Inc., Korea). Arraying was performed into 8 mm diameter wells of 96-well type plate and cured for 16 h for gelation according to manufacturer’s recommendation. The final arrayed sol-gel microdroplet volume was calculated using autodrop volume detection software (Scienion, Germany, http://www.scienion.com). Approximately 6 × 1010 anti-core aptamers and 3 × 1011 #12 aptamers were immobilized in a sol-gel microdroplet and the single microdroplet volume was around 5 and 25 nL, respectively. Measurement of the yTBP Detection Limit. Each well was incubated for 2 h with a blocking buffer (Binding buffer [25 mM Tris, pH 8.0, 100 mM NaCl, 25 mM KCl, and 10 mM MgCl2] with 5% skim milk). Subsequently, yTBP (from 0 to 3.2 µM) and 2 µM of Cy-3 labeled aptamers #24 in 50 µL of binding buffer were added to each microarray well and incubated. The resulting aptamer microarray was scanned and analyzed using a 96-well fluorescence scanner and the appropriate software program (FLA-5100 and multigauge, Fuji, Japan). The background intensity was subtracted from the signal intensity of each spot (LAU/mm2). Tryptic Digestion in Sol-Gels: “In Sol-Gel Digestion”. To optimize the complete trypsin digestion in the 3-dimensional sol-gel microdroplet array, three different tryptic conditions (4, 40, 400 nM in 50 µL of buffer solution) with the controls (without trypsin) were applied to the aptamer microarray. In detail, the wells of aptamer microarray were incubated with 8 µM yTBP and trypsin was then added and incubated to each well for 4 h at 37 °C. After washing 3 times with 0.2% Tween 20 treated binding buffer, each well was incubated with the Cy-3 labeled aptamer #24 and scanned for remaining intact yTBP, determining the optimal tryptic conditions. Peptide Recovery from Aptamer Microarray. Sol-gels containing TBP specific aptamer #12 were arrayed into a 96well type plate as described above in Microarray Preparation. Each well contained 16 sol-gel microdroplets with the 4 × 4 arrays. To retrieve the peptide which can bind to aptamers, yTBP spiked in human serum (20 ng/µL) were incubated in eight identical wells for 1 h. After washing, 400 nM trypsin solution was directly added to each well. To prevent the evaporation during the tryptic digestion, the plate was completely wrapped with plastic-wrap. After digestion, the solutions were retrieved from the 8-wells using a micropipet and dried in a vacuum centrifuge. The peptide extracts were resuspended in 2 µL of 0.1% formic acid, 5% acetonitrile in water and combined all together in one. The total solution was pretreated for desalting using a microcolumn made of a GelLoader tip (Eppendorf, Hamburg, Germany). The loaded peptides were Journal of Proteome Research • Vol. 9, No. 11, 2010 5569

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Figure 1. Schematic diagram of the “Aptamer microarray mediated capture and identification” approach. First, a sol-gel formulation was used to immobilize the aptamers. A large number of active aptamers can be entrapped within the 3-dimensional nanoporous sol-gel matrix. Unlike the antibodies, aptamers, as a capturing agent, do not produce peptides after a digestion step that interferes with proteome data analysis. Therefore, an LC-ESI/MS coupled aptamer microarray can become a new approach for biomarker identification in complex clinical samples.

washed with 5% formic acid, eluted in 1 µL of 50% acetonitrile in deionized water and 0.1% formic acid, dried in a vacuum centrifuge, and dissolved in 4 µL of 0.1% formic acid. Processing for LC-ESI Mass Spectrometry. The desalted peptides were analyzed on an HP 1100 HPLC nanoflow system using a splitter (Agilent) coupled online to an LCQ DECA ion trap (Thermo Fisher Scientific, San Jose, CA). Zorbax 300SBC18 resin (particle size 5 µm, Agilent Technologies) was packed into a home-built fused silica column (100 mm length × 75 µm i.d., tip diameter 10 µm). Peptides were bound in a flow of buffer A (0.1% (v/v) formic acid and 5% acetonitrile in water) for 10 min at 600 nL/min. The gradient was employed with buffer B (0.1% (v/v) formic acid in 95% acetonitrile) at a constant flow rate of 200 nL/min: 5-12% in 10 min, 12-35% in 25 min, 35-90% in 5 min, 90% for 10 min. A 1.7 kV spray voltage was applied at the ESI source and the transfer capillary temperature was set at 180 °C. MS scan events in positive ion mode were controlled by Xcalibur 1.2 software. Precursor ions were selected over the range m/z 350-2000 for MS/MS fragmentation within a (3 m/z window for subsequent MS/ MS scans in a data-dependent mode. An exclusion dynamic mode was applied to exclude the selected most intense ion from further selection over 2 min period. MS/MS data were acquired using a 2 m/z unit ion isolation window in the automated gain control (AGC) mode where AGC values of 5.00 × 105 and 1.00 × 104 were set for full MS and MS/MS, respectively. The normalized CID was set to be 35.0. Peak Analysis for LC-ESI Mass Spectrometry. Peak lists of MS/MS spectra were processed using Analyst QS (v1.1, Applied Biosystems, Foster City, CA) software and searched against the 5570

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NCBI (National Center for Biotechnology Information)-nonredundant (nr) (version 14 Aug 2006, 486 696 mammalian entries) and EST (Expressed Sequence Tag)_yTBP (ACCESSION O43133) database using Mascot19 operating on a local server (version 2.1, Matrix Science). Initial search parameters were the following: allowance of tryptic missed cleavages, 2; variable modification parameters: Carbamidomethyl (C), Deamidation (NQ), Oxidation (M), Propionamide (C); peptide tolerance, 1.0 Da; MS/MS tolerance, 0.8 Da; default charge state, +2 and +3; and asparagine/glutamine (formally ‘deamidation’, but due to deisotoping artifact during data extraction). The candidate peptides, whose probability MOWSE (MOlecular Weight Search) scores are statistically meaningful (p < 0.05), were only selected using MSQuant, version 1.4.16a (www.cebi.sud.dk). Proteins identified with at least two peptides were taken without any manual validation. Protein identifications with a single significant peptide were manually verified by the inspection of the MS/MS fragment ion spectrum. Different isoforms of the protein reported were verified by identification of at least one unique peptide. Only the highest scoring identification was selected where multiple proteins ID were listed.

Results and Discussion We have previously reported three sol-gel formulations designed for biomolecule entrapment, such as antibody, protein and small molecules in sol-gel microdroplets,15 and anti-HCV core aptamer as a sensing material in a chip-based diagnostic tool.18 Figure 1 illustrates the scheme of our approach to identify a target protein using aptamer affinity probes

Target Protein Identification from Aptamer Microarray Platform

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Figure 3. (A) The illustration of the aptamer-target-aptamer sandwich assay platform. (B) After immobilizing the aptamer #12, TBP (3.2 mM) and Cy-3 labeled aptamer (#24, 2 µM) were sequentially incubated in the well. As a negative control, sandwich assay was performed without TBP (see the right well picture). P, Cy3-dUTP as a positive signal reference.

Figure 2. Optimal sol-gel formulation selection for aptamer immobilization. (A) We applied the aptamer-target-antibody sandwich format for selecting the optimal aptamer immobilization in microarray. As shown in the illustration, anti-core aptamers were mixed with three sol-gel formulations (F-I, F-II, and F-III) and were arrayed onto the surface of 96-well plate. Then, target protein (HCV core protein, 20 kDa,) and anti-core antibody sets (anti-HCV core and anti-mouse-Cy3) were sequentially applied to the array. Their binding property was analyzed by fluorescence scanner. (B) The percent formula for F-I, F-II and F-III was described in Materials and Methods and scanned feature of microarray after assay was shown with negative and positive signal reference. [N, negative sol-gel droplet (without aptamer); A, anti-core aptamer, P, Cy3-labeled antibody as a positive signal reference.]

entrapped in sol-gel microdroplets. Yeast TBP (TATA Binding Protein) was chosen as a target protein because novel aptamers with high affinity to TBP have been isolated (unpublished data). We used anti-core HCV aptamers18 for finding optimal conditions for aptamer sol-gel microarray immobilization (Figure 2A). Anti-core HCV aptamers were captured by mixing with three sol-gel formulations (Formulation-I, FormulationII, and Fomulation-III) and were arrayed on the surface of 96well plate. Around 6 × 1010 aptamer molecules can be immobilized in a single droplet and the volume of single microdroplet is ∼5 nL. After target molecule incubation (4 µM of HCV core protein in binding buffer), labeled anti-core antibody was sequentially added to the well. The binding properties were analyzed by a fluorescence scanner. As shown in Figure 2B, only one formulation (F-II) can immobilize aptamers with high

sensitivity among three formulations, and hence, this formulation was used for the immobilization of aptamer #12 in the TBP assay. Formulation F-I shows nonspecific binding both at aptamers and negative control mcirodroplets. Formulation F-III does not show any signal in the aptamer microdroplets. The capturing affinity properties of the aptamer were first tested with a sandwich assay. The appropriate aptamer pair (#12 and #24) was considered for sandwich format (Figure 3A). Aptamer #12 was selected as the capturing affinity agent because it showed the highest binding to yTBP and aptamer #24 was used as the detection probe after being fluorescently labeled. Within each well, 16 (4 × 4 array) duplicate spots, containing the #12 TBP aptamers as a capturing material were arrayed along with positive controls (P; Cy-3 d-UTP). Around 3 × 1011 aptamers were embedded in a single droplet (spot volume is ∼25 nL). After TBP incubation, the other aptamer pair (Cy-3 labeled aptamer #24) was sequentially incubated for 1 h. As shown in Figure 3B, entrapped aptamer #12 can maintain its activity in sol-gel droplets required for capturing their target molecules, TBP proteins, by aptamer-target-aptamer sandwich assay. In contrast, the interaction between aptamers (#12 and #24) without TBP proteins was not observed as shown in the control experiment (Figure 3B, right well). Then, various concentrations of target proteins (yTBP, from 0 to 3.2 µM) were tested to measure the detection limit. In Figure 4, the dynamic range of fluorescent signals is plotted as a function of the yTBP concentration. Since the amount of bound yTBP is influenced by the fluorescently labeled upper aptamer (#24), final signal intensities of resultant microdroplets represent the binding quantities of yTBP. The results showed a dynamic range of close to 3 orders of magnitude (from a few nanomolar to micromolar) with good linearity up to 400 nM. Journal of Proteome Research • Vol. 9, No. 11, 2010 5571

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Figure 5. Yeast TBP sequence and two matched peptides (red bold) of the yeast TATA box protein obtained by LC coupled ESI-MS/MS analysis. Figure 4. The dynamic range of fluorescent signals vs the yTBP concentrations. The capturing aptamer (#12) was embedded in the sol-gel droplets, and then, different concentrations of TBPs were incubated in each well. For detecting the amount of TBP bound to aptamer, Cy-3-labeled aptamers (#24) were added to the aptamer microarray. Measured fluorescence intensities (AU) were plotted versus the TBP concentrations. The dotted line represents the background signal of the well.

This suggests that the sol-gel embedded capturing aptamer (#12) can be used for quantifying the target molecules, indicating that future developments would provide aptamer based microarray MS for quantitative studies of differential disease biomarker expression. Moreover, it has the potential to allow absolute quantification of nanomolar range target molecules in the linear section using the appropriate internal standard. This approach can also lead to the identification of the target protein from crude samples, such as serum. To accomplish this, the targets bound to aptamers should be digested completely inside the sol-gel droplets. Since very large amounts of target analytes may be bound to the aptamers inside the nanoliter scale flat-hemispherical droplets, it is important to search for suitable tryptic conditions for the aptamer microarray. After yTBP binding to the aptamer microarray, each well was incubated with different concentrations of trypsin for 4 h. The amounts of yTBP were estimated using a second aptamer pair, which is analogous to a sandwich assay (see Supporting Information Figure 1). This method is newly called “In Sol-gel digestion” and is different from conventional in-gel digestion method. The optimal reaction conditions were determined by titrating the trypsin concentration, yielding a protocol for “In Sol-gel digestion” condition with 400 nM trypsin. Subsequently, human serum spiked with yTBP was incubated for 1 h, followed by washing. After the tryptic digestion, the digest was loaded into ESI/MS, electrospray mass spectrometry, for subsequent identification of the targeted protein according to frequently reported methodology.20,21 As shown in Figure 5, two matched peptides of the yTBP were identified from database search; the NCBI-nonredundant (nr) (version 14 Aug 2006, 486 696 mammalian entries) and EST_yTBP (ACCESSION O43133) database using Mascot. We compared these obtained peptide results between the NCBI-nonredundant and EST_yTBP database. These peptides were only observed in TBP database. Moreover, other serum proteins were not observed in this proteome analysis, and hence, no background interference was observed from the aptamer capture probes. In summary, the yTBP-aptamer microarray captured and identified the target molecules selectively. Generally, highly 5572

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abundant ‘common housekeeping’ proteins, such as albumin and immunoglobulins, constitute approximately 60-97% of the total serum protein,22 which prevent the detection of lowabundance proteins, some of which might be biomarker proteins. Therefore, additional steps for removing the abundant proteins are needed in whole proteome analysis. In this study, 10 µg of yTBP was mixed with the total serum proteins (concentration of the serum protein was ∼10 000 µg in 500 µL) such that the percentage of yTBP was 0.001%. The lowabundance target was identified from the serum without any pretreatment step. This method holds potential to rapid quantification of well-known biomarkers in blood or urine as well as for biomarker discovery in extracts from cancer cells or tissue biopsies. Recently, high-affinity aptamers against several types of cancer were generated using a whole living CellSELEX process.23-25 These have also been modified with drugs or contrast agents to open a new route to targeted drug delivery or bioimaging studies, respectively. Aptamer microarrays can allow the detection of any type of biomarkers generated by cancer cells and is applicable to a broad range of proteome research.

Acknowledgment. This study was supported by the Korea-Sweden research cooperation program (STINT and KOSEF). The authors acknowledge funding from National Research Laboratory from NRF (National Research Foundation) and Ministry of Knowledge Economy, and Industrial Technology Development (10032113). Ji-Young Ahn would like to acknowledge the support of the Korea Research Foundation Grant (KRF-2008-532-D00003/2009-353-D00004). D.-k.L. acknowledges the support from Global Research Laboratory grant by Korean Ministry of Education, Science and Technology. Supporting Information Available: Tryptic condition for a complete digetion on the sol-gel droplets (In Sol-gel Digestion). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) van Gils, M. P.; Stenman, U. H.; Schalken, J. A.; Schroder, F. H.; Luider, T. M.; Lilja, H.; Bjartell, A.; Hamdy, F. C.; Pettersson, K. S.; Bischoff, R.; Takalo, H.; Nilsson, O.; Mulders, P. F.; Bangma, C. H. Eur. Urol. 2005, 48, 1031–1041. (2) Righetti, P. G.; Boschetti, E.; Lomas, L.; Citterio, A. Proteomics 2006, 6, 3980–3992. (3) Hager, G.; Cacsire-Castillo, D.; Schiebel, I.; Rezniczek, G. A.; Watrowski, R.; Speiser, P.; Zeillinger, R. Gynecol. Oncol. 2005, 98, 211–216.

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Target Protein Identification from Aptamer Microarray Platform (4) Anderson, L.; Hunter, C. L. Mol. Cell. Proteomics 2006, 5, 573– 588. (5) Colzani, M.; Schutz, F.; Potts, A.; Waridel, P.; Quadroni, M. Mol. Cell. Proteomics 2008, 7, 927–937. (6) Comuzzi, B.; Sadar, M. D. Cellscience 2006, 3, 61–81. (7) Liang, X.; Hajivandi, M.; Veach, D.; Wisniewski, D.; Clarkson, B.; Resh, M. D.; Pope, R. M. Proteomics 2006, 6, 4554–4564. (8) Ren, S.; Yoon, H. R.; Kim, S. Y. Biochip J. 2008, 2, 155–159. (9) Ulrich, H. Med. Chem. 2005, 1, 199–208. (10) Shu, D.; Guo, P. J. Biol. Chem. 2003, 278, 7119–7125. (11) Ahn, Y. H.; Lee, J. Y.; Kim, Y. S.; Ko, J. H.; Yoo, J. S. J. Proteome Res. 2009, 8, 4216–4224. (12) Anderson, N. L.; Jackson, A.; Smith, D.; Hardie, D.; Borchers, C.; Pearson, T. W. Mol. Cell. Proteomics 2009, 8, 995–1005. (13) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. Anal. Chem. 2001, 73, 5088–5096. (14) Ekstrom, S.; Onnerfjord, P.; Nilsson, J.; Bengtsson, M.; Laurell, T.; Marko-Varga, G. Anal. Chem. 2000, 72, 286–293. (15) Kim, S.; Kim, Y.; Kim, P.; Ha, J.; Kim, K.; Sohn, M.; Yoo, J. S.; Lee, J.; Kwon, J. A.; Lee, K. N. Anal. Chem. 2006, 78, 7392–7396. (16) Fan, X.; Shi, H.; Adelman, K.; Lis, J. T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6934–6939.

(17) Park, S. M.; Ahn, J. Y.; Jo, M.; Lee, D. K.; Lis, J. T.; Craighead, H. G.; Kim, S. Lab Chip 2009, 9, 1206–1212. (18) Lee, S.; Kim, Y. S.; Jo, M.; Jin, M.; Lee, D. K.; Kim, S. Biochem. Biophys. Res. Commun. 2007, 358, 47–52. (19) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551–3567. (20) Hanas, J. S.; Hocker, J. R.; Cheung, J. Y.; Larabee, J. L.; Lerner, M. R.; Lightfoot, S. A.; Morgan, D. L.; Denson, K. D.; Prejeant, K. C.; Gusev, Y.; Smith, B. J.; Hanas, R. J.; Postier, R. G.; Brackett, D. J. Pancreas 2008, 36, 61–69. (21) Heo, S. H.; Lee, S. J.; Ryoo, H. M.; Park, J. Y.; Cho, J. Y. Proteomics 2007, 7, 4292–4302. (22) Georgiou, H. M.; Rice, G. E.; Baker, M. S. Proteomics 2001, 1, 1503– 1506. (23) Cerchia, L.; Giangrande, P. H.; McNamara, J. O.; de Franciscis, V. Methods Mol. Biol. 2009, 535, 59–78. (24) Guo, K. T.; Paul, A.; Schichor, C.; Ziemer, G.; Wendel, H. P. Int. J. Mol. Sci. 2008, 9, 668–678. (25) Shangguan, D.; Cao, Z.; Meng, L.; Mallikaratchy, P.; Sefah, K.; Wang, H.; Li, Y.; Tan, W. J. Proteome Res. 2008, 7, 2133–2139.

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