Proteochip: a Highly Sensitive Protein Microarray Prepared by a Novel

Proteomics 2003, 3, 2289–2304 DOI 10.1002/pmic.200300541 2289 Yoonsuk Lee1 ProteoChip: A highly sensitive protein microarray Eun Kyoung Lee1 Yong Wan Cho1 prepared by a novel method of protein Takuya Matsui2 immobilization for application of protein-protein In-Cheol Kang3 Tai-Sun Kim4 interaction studies Moon Hi Han1 We have developed a highly sensitive microarray protein chip, ProteoChip, coated with 1 Proteogen, ProLinker, novel calixcrown derivatives with a bifunctional coupling property that per- Seoul, Korea mits efficient immobilization of capture proteins on solid matrixes and makes high- 2Bio-Medical Center, Naka Division, throughput analysis of protein-protein interactions possible. The analysis of quartz Hitachi High-Technologies, crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) Hitachinaka-shi, bound to the gold film of the sensor surface coated with ProLinker B and that it is Ibaraki-ken, Japan useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with 3Protein Chip Research Center, ProLinker A, was also demonstrated to be useful for preparation of high-density array Biotechnology Research Institute, spots by using a microarrayer and for analysis of analyte Ags either by direct or sand- Cungbuk National University, wich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip Cheongju, Korea was as low as 1–10 femtogram/mL of analyte protein, useful for detection of tumor 4Department of Chemistry, markers. ProteoChip was also useful for studies of direct protein-protein interactions Hallym University, Chuncheon, Korea as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity. Keywords: ProLinker / Protein immobilization / Protein microarray / Protein-protein interaction / ProteoChip PRO 0541 1 Introduction applied for global analysis of expression profiles of proteins [7–9]. Different types of protein or peptide micro- The protein microarray, a solid phase assay method to arrays have been reported to be useful for immuno- detect protein-protein interactions, is becoming an attrac- assays as well as for analyzes of enzymatic activity such tive tool in proteomics today for diagnostics and thera- as kinase activity [10–12]. peutic purposes as well as for basic research [1]. Array- based methods to study proteins allow high-throughput The major advantages of protein array technologies determination of protein functions in parallel [2]. Ekins include (1) being a highly parallel and miniaturized solid and coworkers [3, 5] demonstrated, using the ambient phase assay system, (2) high detection sensitivity, (3) use- analyte assay theory, that a miniaturized binding assay ful for very high-throughput approaches, (4) low con- system, like the microarray, can be highly sensitive. Re- sumptions of reagent samples (nL level), (5) potentially cently, MacBeath and Schreiber [6] reported that a pro- attractive manufacturing costs, and (6) they require only tein microarray chip prepared with aldehyde slides was a few nanograms of protein. The core technologies for useful for studying protein-protein interactions with sev- protein microarrays that are currently practiced by protein eral different protein systems. It was also demonstrated chip developers are surface chemistry for immobilization that a high-density antibody (Ab) microarray could be of proteins or capture agents, capture molecules which are immobilized onto a solid support and used for captur- Correspondence: Dr. Moon Hi Han, Proteogen, RM 1516 Wind- ing target proteins or molecules, and systems to detect stone, Yangjae-dong, Seocho-ku, Seoul, Korea protein-protein interactions based on fluorescence, che- E-mail: [email protected] miluminescence, MS, electrochemical or surface plas- Fax: 182-2-529-9950 mon resonance (SPR) [9]. Currently, the SPR detection Abbreviations: Ab, antibody; AFM, atomic force microscopy; system, which requires immobilization of capture proteins Ag, antigen; -HCG, beta-human choryonic gonadotropin; â on gold substrate, has been extensively studied and CEA, carcinoembryonic antigen; CRP, C-reactive protein; ECM, extracellular matrix; QCM, quartz crystal microbalance; SAM, widely applied for analyses of protein-protein interactions self-assembled monolayer; SPR, surface plasmon resonance [13–15]. Since a SPR sensor system does not require 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de 2290 Y. Lee et al. Proteomics 2003, 3, 2289–2304 labeling of analyte proteins, it has been recognized to be a substrates for protein immobilization [29–31]. Covalent promising tool for proteomics research. Recently, it was coupling methods allow robust protein immobilization reported that a peptide microarray on a gold-coated sur- with good reproducibility. They can be applied to a wide face can be coupled with various downstream detection range of proteins and versatile linkage processes. How- methods, such as SPR [12]. ever, chemical modification of proteins causes loss of activity and low immobilization efficiency. It also requires The development of a protein microarray, with improved multiple functional surfaces. Abs immobilized on alde- methods of protein immobilization on solid matrix, is of hyde-surfaced solid support had lower binding affinities or great interest to the field of biotechnology [16, 17]. Unlike reduced specificities of Ab-antigen (Ag) interactions [32]. DNA, proteins are chemically and physically heteroge- neous, unstable, have a three-dimensional structure that A specific biomolecular affinity interaction such as the is critical to their function, and can not be amplified [18]. streptavidine-biotin interaction has been widely used as Therefore, development of unique surface chemistry is a biological capture method [25, 26]. The major problem needed for protein microarrays, a challenge that is not with this method is reported to be that biotinylation of Ab present in DNA microarray applications. For miniaturizing molecules causes changes in specificity and affinity of the protein microarray devices, surface chemistry plays an capture Ab [27]. Biological capture methods employing increasingly important role for maintaining the conforma- protein tags provide a stable linkage and bind protein tion and orientation of proteins. An efficient immobilization specifically in a reproducible orientation. However, it method is required for attachment of a wide range of requires biological reagents to be tagged to a target pro- proteins to prevent loss of their activity when bound to the tein adequately and special handling for the preparation surface of solid substrates [19]. Immobilization methods of arrays, and often results in variable stability. using efficient surface chemistry should be reproducible, applicable to proteins with different properties, amenable The decreases in activity of proteins captured on solid to high-throughput automation, and compatible with re- matrixes may be due to the random orientation of capture tention of fully functional protein activity through mainte- proteins, which causes decreased accessibility for inter- nance of the correct orientation of surface-bound proteins, acting proteins, or conformational changes in a protein and minimization of nonspecific protein binding [18, 20]. especially around its active site due to tight binding to the chip surface [33, 34]. Furthermore, the immobilization Several different immobilization methods and tags have process of a capture protein should be simple and easy been described for construction of protein microarrays. for microarray preparation for high-throughput applica- Protein immobilization has been carried out by applying tions. Improved surface chemistry for immobilization of various methods, such as noncovalent absorption [21, capture proteins, to preserve their activity and maintain a 22], covalent linkage by chemical bonding [23, 24], and correct orientation, has been suggested to be necessary affinity capture methods such as the streptavidine-biotin for development of high quality protein microarrays. interaction [25–27]. There are both advantages and disad- vantages to these methods of protein immobilization. We present a novel immobilization method to capture Noncovalent binding of proteins has the advantage of proteins which uses calixcrown derivatives (ProLinker A low nonspecific binding to glass surface and high ca- and B) on solid substrates. It provides effective high-den- pacity and retention of protein function [20]. The method sity protein immobilization without activity loss or incor- of noncovalent physical adsorption to solid surfaces is rect orientation of the capture protein. The ProteoChip is simple and allows conformation of immobilized protein a useful tool for analysis of protein-protein or Ab-Ag inter- to remain intact. In particular, a hydrogen method appears actions as demonstrated by several experimental results to be useful for enzyme assays. However, these methods with Quartz crystal microbalance (QCM) or fluorescence suffer from some key limitations such as little control immunoassay methods. A mechanism of protein binding of orientation or quantitative adsorption of biomolecules to solid surface coated with ProLinker which results in and,

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