Báo cáo khoa học: Advances in functional protein microarray technology Paul Bertone and Michael Snyder

Advances in Functional Protein Microarray Technology

Tác giả: Paul Bertone and Michael Snyder

Lĩnh vực: Molecular, Cellular and Developmental Biology

Nội dung tài liệu:

Bài viết đánh giá sự tiến bộ trong công nghệ protein microarray chức năng, một phương pháp cho phép quan sát quy mô lớn các hoạt động sinh hóa. Công nghệ này đã phát triển đáng kể nhờ những đổi mới trong sản xuất protein hiệu suất cao và công nghệ bề mặt microarray, cho phép tạo ra các định dạng địa chỉ hóa với mật độ không gian cao. Các ứng dụng tiềm năng bao gồm nghiên cứu sinh học phân tử cơ bản, xác định dấu ấn bệnh, phân tích phản ứng độc hại và sàng lọc mục tiêu dược phẩm. Bài viết cũng đề cập đến những thách thức và cơ hội trong việc phát triển các mảng protein chức năng, bao gồm việc xây dựng thư viện biểu hiện toàn diện, sản xuất protein hiệu suất cao và sự ổn định của protein được sắp xếp. Các phương pháp khác nhau để phát hiện tương tác sinh hóa và các ứng dụng mới của mảng protein cũng được thảo luận.

Mục lục chi tiết:

• MINIREVIEW
• Advances in functional protein microarray technology
• Keywords
• Correspondence
• Abbreviations
• Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, protein-nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening.
• DNA microarrays have become ubiquitous in genomic research, evident by their widespread use in profiling gene expression patterns, mapping novel transcripts, detecting sequence mutations and deletions, and locating transcription factor binding sites. Although microarray experiments are invaluable for large-scale sequence analyses, little can be inferred from these studies about the functions of gene products. In contrast to the high-throughput (HT) experiments afforded by DNA arrays, those designed to elucidate the biochemical activities of encoded proteins have traditionally been carried out on single molecules. Recently, significant effort has been made towards adapting proteomic analytical methods for use with DNA microarray technologies to enable the elucidation of proteome-wide biochemical activities and interactions.
• The large-scale characterization of protein complexes generally involves (a) the separation of complex protein samples and (b) the subsequent identification of individual proteins. Among the methods currently available for proteome analysis are 1D gel electrophoresis (1D-GE) and 2D gel electrophoresis (2D-GE), MS, affinity chromatography, N-terminal Edman protein sequencing, metal affinity shift assays, 15N isotope labeling, and tandem affinity purification (TAP) tagging [1] to isolate protein complexes from cell extracts. Of these, protein separation by 2D-GE and subsequent identification using MS have remained the two core technologies for large-scale proteomics. The 2D-GE method entails the separation of complex protein mixtures by molecular charge in the first dimension and by mass in the second dimension. Although recent advances in 2D-GE have improved the resolution and reproducibility, the technique remains difficult to automate in a HT setting. For this reason, alternative approaches that obviate the need for gel separation, such as multidimensional protein identification technology [2], have gained popularity for large-scale proteomics efforts and are able to generate a comprehensive catalog of proteins present in complex cell extracts. HT protein analysis is expected to accelerate with the introduction of new robotic liquid
• chromatography systems and high-resolution analysis methods such as top-down Fourier transform mass spectrometry [3].
• The use of MS for protein identification has come into wider use with the advent of soft ionization techniques, such as electrospray ionization (ESI) [4] and MALDI [5,6]. Additionally, the emergence of hybrid methods incorporating electrospray technology [7] with quadrupole time-of-flight mass spectrometer tandem mass analysis (ESI Q-TOF MS/MS) allows more accurate identification of specific proteins through the generation of collision-induced dissociation (CID) spectra that yield accurate sequence tags from protonated peptide ions. Recently, the range of mass spectrometric applications has been extended by other tandem approaches such as as MALDI TOF-TOF MS/MS [8,9] and MALDI Q-TOF MS/MS [10].
• Beyond the identification of individual proteins, quantitative analysis of complex samples can be accomplished through the use of surface-enhanced laser desorption/ionization (SELDI) [11,12]. This approach incorporates standard ionization techniques on different surfaces, comprising a solid support modified with various chemical or biological bait molecules. These may include antibodies, proteins, nucleic acids and metal ions. The differential surface capture of solubilized protein samples provides a unique signature that varies depending on protein composition. Unlike MS techniques, SELDI is not able to identify specific proteins in a complex sample. This is expected to change in the near future through the combined use of SELDI technology with tandem mass spectrometers.
• Regardless of the methods used to measure and catalog an organism’s proteome, the majority of detection and quantification methods result in denaturing of the protein samples and thus functional characterization is not possible. To obtain detailed functional information, proteins must be cloned and expressed in recombinant form and subjected to systematic biochemical analyses. Martzen et al. [13] developed a multiplexed assay to characterize protein function on a large scale through a divide-and-conquer strategy. The approach entails the generation of pooled purified protein samples, which are then assayed in parallel for various biochemical activities. Individual proteins that exhibit specific activities in a pooled sample are identified through a series of recurrent analyses of subpools. This procedure allows the rapid identification of proteins that participate in various biochemical pathways via a divisive search through subpopulations of functional proteins. An advantage of this method is that biochemically active, multimeric protein complexes may be identified in vitro; however, this does not represent an exhaustive combinatorial search as only proteins that happen to be present in a given pool may interact.
• Development of addressable protein arrays
• The ultimate goal of protein microarray development is to construct ordered arrays of individual proteins to assess biochemical activities on a single-molecule basis. One solution has entailed the use of analytical arrays for the purpose of protein profiling. These typically comprise a library of peptides or antibodies arrayed on the surface of a glass microscope slide [14]. In general, protein profiling entails the measurement of binding specificity, affinity, or abundance of proteins in a biological sample. To address this with microarrays involves the construction of an array whose elements are designed to capture, and thereby measure the binding specificity of, proteins present in a complex mixture such as a cell lysate or serum sample. Array features are typically antibodies that are mechanically printed as independent purified samples, but may also be peptides that are synthesized in situ. The latter technology holds promise for the rapid screening of high-affinity binding sequences and the identification of potential drug targets.
• The synthesis of individual peptides in situ can be accomplished via photolithography, originally described by Fodor et al. [15] and later applied to oligonucleotide-based microarray fabrication [16]. Photolithographic peptide arrays involve the use of photolabile phosphoramitides that enable deprotection of the Boc group from an amino moiety, allowing polymer synthesis to proceed at discrete array locations when illuminated by a laser. Using this method, an array of 1024 peptides was constructed and probed with a mAb. Pellois et al. [17] developed an alternative technique that can make use of natural amino acids for peptide synthesis, using a photogenerated acid to chemically deprotect the growing polymer chain upon exposure to light.
• A simpler approach to photolithography is described by the SPOT protocol [18], in which a series of activated amino acids is mechanically deposited onto a porous surface, thereby building the desired peptides sequentially. SPOT is based on conventional solid-phase synthesis chemistry, and may therefore be more accessible in terms of implementation.
• Recently, Li et al. [19] described a novel approach to homogeneous in situ peptide synthesis based on a common cyclic peptide scaffold. The procedure involves the deprotection of NPPOC phosphoramitide
• groups to affect the addition of side-chains to a universal core molecule, which is presythesized and applied to a silanized glass slide in a uniform manner. A library of individual peptides can then be synthesized in situ using maskless photolithography [20], in which a spatially addressable array is fabricated through successive photodeprotection using a bank of digitally controlled micromirrors.
• To date, most protein microarray systems have been based on contact-printed antibody libraries that are used for profiling complex analyte mixtures. The most widely adopted strategy consists of a multiplex adaptation of the classical antibody sandwich assay, where a pair of antibodies binds two discrete recognition surfaces on each protein [21,22]. In this procedure, an array of antibodies, which have been immobilized through covalent bonding to a silanized glass surface, is probed with various analytes. A second biotinylated antibody is then applied which binds to captured analytes, forming an immune complex. The second antibody is finally detected with a universal antibiotin antibody conjugated to a fluorophore. This approach has been used to great effect for the simultaneous detection of multiple cytokine or chemokine levels in biological samples [23]. The highly specific antibody-complex recognition is ideal for detecting low-abundance cytokines and holds much potential for clinical diagnostic applications and discovery of therapeutic drug targets [24].
• A variation on this experiment utilizes rolling circle amplification (RCA) to enhance the fluorescence signals emitted from the immune complex [25]. In this method, the detection antibody is not fluorescence-labeled but is instead conjugated to oligonucleotides that serve to prime the RCA reaction. Complementary circular oligonucleotides are extended with DNA polymerase, producing RCA products that consist of tandem repeats. Because these repeat sequences provide many redundant hybridization targets, an amplification in fluorescence signal is achieved when the RCA products are detected with fluorescence-labeled complementary DNA probes.
• An impediment to the further development of antibody array technology lies in the availability of high-quality antibodies against the individual proteins in a complex sample. At present it remains unfeasible to obtain hundreds or thousands of different antibodies that can recognize and capture various proteins with high affinity and specificity – factors that are essential for preventing cross-reactivity. The problem is compounded when considering multiplex sandwich immunoassays, where two highly specific antibodies must be obtained for each protein captured. These must also recognize two different regions of the protein, each without masking the other binding domain.
• Although this issue may eventually be addressed by new methods of HT antibody generation, several alternatives to protein capture have been developed that do not rely on antibody recognition. Among the most innovative of these involves imprinting technology to create artificial molecular recognition surfaces [26,27]. Peptides that correspond to signal sequences in various target proteins are used as a structural scaffold, around which polymerizable monomers are allowed to self-assemble. The monomers are crosslinked in place and the template molecule is stably removed from the complex. The cavity or imprint that remains is shape-complementary to the original template and will therefore bind identical structures with high affinity. This technology promises to accelerate antibody array development by increasing the throughput of artificial epitope production, although at present imprinting is unable to mimic larger functional proteins or other macromolecular structures.
• The antibody array represents an excellent platform for HT protein profiling. However, the large-scale study of protein biochemistry using the microarray format requires the development of arrays of full-length, functional proteins. HT protein production, combined with technologies shared with the proven DNA microarray format, allow the simultaneous analysis of thousands of protein activities in a single experiment. In addition to enabling the inverse profiling experiment where functional proteins can be interrogated with individual antibodies [28], protein microarrays enable a wide range of biochemical assays in response to any solution-phase binder.
• Array technologies for functional protein analysis
• The principal challenges in functional protein array development comprise (a) creation of a comprehensive expression clone library, (b) HT protein production, including expression, isolation and purification, (c) adaptation of DNA microarray technology to accommodate protein substrates, (d) ensuring the stability of arrayed proteins, and (e) reduction of inter- and intra-slide variability of protein concentration between deposited samples.
• By far the greatest obstacle in developing functional protein microarrays is the construction of a comprehensive expression clone library from which a large number of distinct protein samples can be produced (Fig. 1). In building a clone library, it is desirable to construct recombinant genes where fusion proteins can be produced for the purpose of affinity purification and/or slide surface attachment. Cloning the genes of
• interest with an inducible promoter allows individual proteins to be expressed in high abundance. HT purification can be accomplished with the addition of C- or N-terminal tags, such as glutathione-S-transferase or the IgG-binding domain of Protein A. The incorporation of fusion tags also facilitates the verification of clone inserts by sequencing across the vector-insert junction. It is highly desirable to transform the expression vector into a homologous or related cell type, ensuring the proper delivery of the protein product to the secretory pathway and hence correct folding and post-translational modification of each recombinant protein.
• Prototype formats for functional protein arrays
• A critical aspect of the development of arrays of functional proteins is the selection of an experimental support and its associated method of surface attachment and immobilization of proteins. If proteins can be immobilized without disrupting their native conformations, they are likely to remain biologically active in vitro. The method of immobilization will also greatly influence the orientation in which proteins are attached to the support surface. Engineering a common point of attachment for all samples, typically through the inclusion of affinity tags, ensures that at least a subset of proteins maintain a uniform presentation to solution-phase binders. The array format must be compatible with appropriate detection instruments and exhibit a wide dynamic range of intensity values associated with protein binding or catalysis events. Additionally, nonspecific binding of labeled samples should be minimized in order to reduce the background and increase the signal-to-noise ratio of the experimental platform.
• The materials traditionally associated with conventional protein assays are often incompatible with robotic arrayers, cannot provide the sensitivity or dynamic range expected from microarray experiments, or contribute high fluorescence background resulting in low signal-to-noise ratios. To circumvent these problems, a number of innovative platforms have been explored for prototyping the protein array format. Among these, two make use of an agarose or acrylamide gel situated on glass slides, thereby combining the utility of a solid support with the loading and binding capacity of a porous gel matrix. These methods were originally devised to increase the potential loading capacity of DNA samples on a planar microarray surface, but are generally applicable to a variety of substrates, including nucleic acids, proteins and small molecules.
• Guschin et al. [29] developed arrays of polyacrylamide gel pads on a hydrophobic glass surface using a combination of gel photopolymerization and manual contact pin deposition. Three test proteins were loaded onto the arrays mouse IgG1, rabbit IgG and BSA. Immunoanalysis of fluorescein isothiocyanate
• (FITC)-labeled IgG against mouse IgG1 demonstrated selective binding when the arrays were imaged with a fluorescence microscope. In a related study, Mirzabekov and coworkers [30,31] experimented with a copolymerized acrylamide-bisacrylamide substrate, producing arrays of discrete gel pads of between 10 and 100 µm in diameter. In a later study, Kiyonaka et al. [32] developed a method of supramolecular gel formation as a spontaneous process that does not require additional polymerization steps, using the approach to develop a sensor array of fluorescent metal anion and cation receptors in a glycosylated amino acetate hydrogel matrix [33].
• A study by Afanassiev et al. [34] explored the use of a thin, uniform layer of agarose film on a glass surface to achieve a similar effect. Activated agarose containing NaIO4 was applied to silanized slides, and samples were mechanically deposited using a robotic microspotter after the gel had solidified. A limited application of this platform to protein-based assays demonstrated the binding of mAbs to immobilized recombinant human BAD protein, and reciprocally, of recombinant human (rh)BAD to immobilized antibodies in a sandwich immunoassay. Another approach involved the use of a liquid silica compound to create flexible sheets of microwell arrays in which biochemical reactions are performed en masse. Zhu et al. [35] devised a system of casting a silicone elastomer (polydimethylsiloxane) onto a reusable mold of laser-milled acrylic. After the microwell sheets had cured, various molecules were immobilized to the interior surface of the wells using the chemical crosslinking agent, 3-glycidoxypropyltrimethoxysilane.
• Aside from demonstrating the technical feasibility of protein immobilization and binding to various molecules in vitro, it is essential to conduct biologically relevant experiments if protein microarrays are to become an established research platform. To explore the utility of microwell arrays for the detection of enzymatic activities, Zhu et al. [35] focused on 119 protein kinases from the budding yeast Saccharomyces cerevisiae (95 known and 24 uncharacterized). Each of the protein kinases was expressed in recombinant form and purified, then assayed for the ability to phosphorylate 17 substrate proteins. A total of 17 arrays were fabricated and one of the substrate proteins was immobilized in every well; a different protein kinase was delivered into each well in the presence of y-ATP to determine which kinases were capable of phosphorylating the given substrate protein. The arrays were incubated and then washed such that all free enzyme was removed, and the signal from the radiolabeled proteins in each well were quantified using a phosphoimager. The
• possibility that measurements originated from the autophosphorylation of the kinases themselves was discounted, as in each case the substrates were bound in the wells while the enzymes remained free in solution; the wells were cleared of this reaction mixture and washed prior to imaging.
• In addition to detecting expected phosphorylation activities, 27 protein kinases were found to be capable of phosphorylating tyrosine after incubation with a synthesized poly(tyrosine-glutamate) peptide. This finding was significant as yeast protein kinases are generally known to phosphorylate only serine or threonine. Phylogenetic analysis revealed sequence similarities among the tyrosine-phosphorylating kinases that were specific to several amino acids oriented in the catalytic cleft and substrate-binding domain of the enzyme. These appeared exclusively in the kinases that phosphorylated tyrosine and not in those that phosphorylated serine or threonine alone. Although it is likely that most yeast protein kinases will preferentially phosphorylate serine or threonine in vivo, this study demonstrated that protein arrays are sensitive enough to reveal previously uncharacterized biochemical properties in a HT assay.
• On balance, each of these formats retains a number of important properties for proteomic experiments. Microwells provide the ability to preserve native protein function by carrying out reactions