Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

8-nitronaphtylethyloxycarbonyl, DMBoc: 5’-O-dimethoxybenzoincarbonyl, NPPoc: 2-(2-nitrophenyl)- propylxycarbonyl, PYMoc: pyrenylmethyloxycarbonyl.

mask [152], to facilitate the in situ synthesis of peptide [128] or oligonucleotide [87, 42] microarrays on glass surfaces.

8.4.3. CD, Microfluidics, Fiber Optic Microarray, Multiplex Beads

The compact disc-based microarray system was developed by Kido et al. and used for immunoassays [67]. They used the piezoelectric inkjet applicator to deposit the proteins onto a polycarbonate disc. Recently, Clair et al. reported the attachment of small molecules to a polycarbonate compact disc (CD) surface via phosphodiester bond. Molecular interactions between analyte and some of these molecules could be detected with a conventional CD player [76]. Walt et al. developed the fiber-optic microarray biosensor technology that has been commercialized, and is now referred as the Illumina BeadArray. This microarray is composed of bundles of selectively etched glass fibers which are dipped into the OBOC oligonucleotide library of microspheres (3 or 5 µm). For some reason, these microspheres are captured randomly and spontaneously at the end of each optical fiber [29, 35, 108, 163]. The array is reusable, allows a rapid response, and has an extremely low detection limits. However, because the microarray is spatially non-addressable, an encoding method is needed. The earlier encoding method uses unique ratio of orthogonal fluorescent dyes to encode the oligonucleotides on each bead [35, 108, 109]. An alternative decoding method uses a unique sequence on each bead of the randomly formed microsphere array as an address. To determine the structure, the microsphere bound oligonucleotide is hybridized to a series of biotinylated complementary oligonucleotides and subsequently visualized by interactions with labeled streptavidin [179]. Epstein et al. described a similar encoding methodology using the displayed oligonucleotide itself to identify its sequence. But, to determine the sequence, the arrays are hybridized to a series of combinatorial decoding

libraries. Four orthogonally labeled decoding libraries are used for each position whereas the screened position in the library is not randomized therefore the affinity of one library to particular bead is higher hence the identity can be determined from level of fluorescence [29]. Recently, encoded fiber-optic microsphere arrays of carbohydrates have been reported [1]. The saccharides were prepared with thiol-terminated ethylenedioxy linker attached to the anomeric center and chemoselectively ligated to commercially available maleimideactivated bovine serum albumin (BSA). The saccharide-BSA conjugate was then coupled to solid support by using water soluble carbodiimide. The BSA molecule serves here as a spacer between the solid support and carbohydrates. The above-mentioned fluorescent dye spectral signature method was used to encode such bead libraries.

8.5. DETECTION METHODS IN CHEMICAL MICROARRAYS

Several methods have been developed for the detection of interactions between the immobilized molecules on the microarrays and the added complex biological mixtures, or analytes. These methods can be broadly classified into two categories: (i) those that involve the detection, identification and characterization of biomolecules (e.g., proteins) present in the analytes that bind to or covalently link to individual chemical spots on the microarrays, and (ii) those that involve the detection and identification of the individual chemical spot that have been enzymatically modified by the enzyme(s), or chemically modified by component(s) present in the analytes.

8.5.1. Identification and Characterization of Bound Proteins

Proteins can bind to peptides or small molecules either through covalent, or noncovalent interactions such as hydrophobic interaction, Van der Waals forces, salt bridges, and hydrogen bonds etc. Common detection methods for protein binding include enzyme-linked colorimetric, fluorescence, luminescence, and radioisotope methods. The fluorescence method is often preferred because it is simple, safe, extremely sensitive, and compatible with the commercially available microarray scanners. The most widely used fluorescent techniques include laser-induced fluorescence, fluorescence resonance energy transfer (FRET), fluorescence polarization, homogenous time resolved fluorescence, and fluorescence correlation spectroscopy [65]. In the standard fluorescence methods, analytes can be directly labeled with fluorescent probe or indirectly with biotin, followed by fluorescent-labeled streptavidin. However, most of the chemical derivatization methods are not site-specific and there is always a concern that chemical labeling of a protein may negatively affect its binding activity to the ligand. An alternative method to fluorescently label a protein is to construct and express the protein as either a fluorophore-puromycin conjugate or red/green fluorescent fusion protein, which can be detected by a conventional fluorescence slide scanner [65, 74]. These methods, while useful, require additional steps, work in some assay systems, but do not work in all proteins. Therefore, alternative methods to detect the bound proteins in their native form are sometimes preferred. One approach is to use fluorescent labeled-antibodies to detect specific proteins bound to the microarrays.

Label-free optical techniques for detecting bound proteins on microarrays have been recently reviewed [23, 90, 162]. These methods include surface plasmon resonance (SPR) [62], grating couplers [13, 112, 169] and reflectometry [18]. SPR has now matured as a

versatile detection tool to analyze the kinetics of protein-ligand interactions over a wide range of molecular weights, affinities, and binding rates [104, 113]. Houseman and Mrksich reported the use of SPR as a detection method to profile carbohydrate-lectin interactions on a carbohydrate arrays [54]. As an alternative detection method, Sapsford et al. used a planar waveguide to develop an antibody array biosensor and studied the kinetics of antigenantibody interactions in parallel [146]. The use of atomic force microscopy (AFM) method to detect the surface topological changes of the microarrays due to bound proteins has been reported [61]. However, AFM is very slow, and the method is impractical unless the chip can be scanned with a large array of AFM probes in parallel.

In recent years, various mass spectrometry technologies have evolved as the dominant tools for identification and characterization of bound proteins [36]. Surface-enhanced laser desorption/ionization (SELDI) mass spectrometry has been developed to detect proteins captured by the very low-density affinity arrays [166]. In this method, captured proteins bound to the metal surface (SELDI protein array) are vaporized using a laser beam, followed by identification of these proteins by mass spectrometry. The detection of a minute amount of bound proteins on high density microarrays by this approach, however, remain to be developed.

8.5.2. Detection Methods to Identify Post-Translational Modification of Proteins or to Quantitate Enzyme Activity in Analytes

Peptide microarrays have been used successfully to probe the activities of specific post-translational modification of enzymes (e.g., proteases, protein kinases, esterases, glycosyl transferases, and acetylase) present in an analyte. Modification of protein, peptide or small molecule spots by these enzymes can be detected by lectins, antibodies, fluorescent or radioactive probes [31, 141, 182]. Zhu et al. analyzed the kinase-substrate specificity of almost all (119 of 122) yeast kinases using 17 different protein substrates [183]. The substrates were first covalently immobilized on the surface of individual nanowells, and individual protein kinases in kinase buffer with [γ33P] ATP were incubated with the substrates. After washing, the nanowell chips were analyzed for 33P-labeled substrates using a phosphoimager (Molecular Dynamics, Inc.) [183]. Recently, a small molecule fluorophore phosphosensor technology referred as Pro-Q Diamond dye has been developed to detect and quantitate phosphorylated amino acids of peptides and proteins in microarrays [103]. To determine the protease substrate specificity, several groups have developed fluorescentquenching methods [20, 143, 185]. These methods are very similar to the on-bead assay for OBOC libraries developed by Meldal’s group described earlier [106]. In this method, quenched fluorescent substrates were prepared by coupling the peptide substrate to coumarin or 2-aminobenzoic acid. These peptide substrates were then spotted onto the solid support and incubated with proteases such as caspase, MMP-2, and trypsin. Peptides that were susceptible to proteolysis fluoresced while others did not.

8.6. APPLICATION OF CHEMICAL MICROARRAY

Chemical genomics is a highly interdisciplinary approach that integrates chemistry and cell biology [157, 158]. Chemical microarray represents an important tool to exploit this emerging field. Chemical microarrays allow investigators to perform many

different assays in parallel using minimal amounts of analytes. Large numbers of biomolecular interactions like protein-protein, protein-ligand, protein-lipid, protein-carbohydrate, and peptide/small molecule-DNA interactions and post-translational modifications can be studied simultaneously by this approach. Information obtained from such studies will facilitate our understanding of cell signaling and function. Like DNA microarrays, the overall pattern of interactions between the analyte and a large number of chemical spots is very informative. Such interaction profiles will allow the investigator to generate conclusions that otherwise would not be possible by only examining a limited number of molecular interactions. Similar to DNA microarrays, bioinformatics and related analytical tools are needed to successfully analyze the data obtained from chemical microarrays. Various aspects of protein microarray technology and antibody microarrays have been recently reviewed, and will only be briefly discussed here [47, 98, 160, 182, 184]. Below is a description on the various biological applications of chemical microarrays, with focus on peptide and small molecule microarrays.

8.6.1. Protein Binding Studies

Protein chips can be used to study protein-protein, protein-nucleic acid, protein-small molecule, and protein-drug interactions [53]. However protein production, purification, and stability are some of the major limiting factors in functional protein microarrays. Proteinprotein interactions often occur between specific protein domains that involve short peptides. For example the SH2 domain binds to a phosphotyrosyl peptide, the SH3 domain binds to a polyproline helix, and integrins bind short peptides such as Arg-Gly-Asp. Therefore, peptide microarrays are useful tools to study protein-protein interactions. Peptides, unlike proteins, are relatively stable and can be readily synthesized by standard solid phase peptide synthesis methods [131]. Espejo et al. reported the development of peptide microarrays with peptide ligands to various known regulatory domain of proteins, such as SH2, SH3, PH, EVHI, PZ and WW. By incubating the peptide microarrays with whole cell lysates, they were able to identify new signaling and associated proteins [6, 30].

Peptide arrays have been used successfully by many investigators for B-cell epitope mapping. As early as 1984, Geysen used multipin technology (peptides immobilized on pins in a 96-well footprint) to map epitopes for monoclonal antibodies [45]. Frank et al. used SPOT synthesis method to prepare a peptide array on a cellulose membrane for epitope mapping [40]. Similarly, the initial application of the first light-directed synthesis of a highdensity peptide microarray was to map the B-cell epitope of anti-β endorphin monoclonal antibody [38].

Recently, Frank and his coworkers have proposed the use of low-density peptide microarrays, which are in situ synthesized on membrane by SPOT synthesis, to pan a phagedisplay protein library that is derived from a randomly fragmented and cloned cDNA library [12]. After affinity enrichment, peptide specific phage populations will be eluted, propagated, labeled, and the identity of the displayed protein determined. This approach is similar to the approach that we have used for studying interactions between OBOC combinatorial small molecule libraries and whole cell extracts, in which a library of the immobilized compounds were screened against a library of target proteins [82]. Reuter et al. reported the use of a peptide array to identify the binding sites of DNA to endonuclease EcoRII. In this study, [32P]-labeled DNA was used to probe a peptide array derived from endonuclease EcoRII sequence [137].

Although there are many reports on using peptide microarrays to probe interactions between peptides and cellular proteins, there have been a limited number of reports on the use of small molecule microarrays to probe cellular proteins. Barnes-Seeman et al. described the identification of new calmodulin-binding small molecules by screening a 6336-member phenol containing fused heterocyclic molecule microarrays [9]. Winssinger et al. used spatially addressable small molecule arrays to study the activity-based profile of proteases in crude cell extracts. In this method a small molecule was covalently tethered to a peptide nucleic acid (PNA) tag, whose sequence, when hybridized to an oligonucleotide microarray, could be used to decode the chemical identity of the small molecule. Using this method, they were able to isolate a small molecule that bound to caspase-3 [171].

8.6.2. Post-Translational Modification, Enzyme-Substrate and Inhibitor Studies

Some of the most important post-translational modifications in cell regulations include phosphorylation, glycosylation, acetylation, and proteolysis. Protein microarrays can be used to identify native substrates for such post-translational modifications. As indicated earlier, protein spots modified by the enzymes can be detected by a radiolabeled substrate (e.g. [γ32P] ATP for protein kinases) and by antibodies or lectins against specific post-translational sites. Zhu et al. analyzed the ability of 119 different yeast kinases to phosphorylate 17 different proteins. They found that members of the yeast Ser-Thr family protein kinases were capable of phosphorylating tyrosine residues of some their substrates [183]. Peptide microarrays have also been a useful tool to study protein phosphorylation. For example, about 10 years ago, we reported on the use of a random OBOC combinatorial library to identify substrate motifs for protein kinase A and c-src protein tyrosine kinase [85, 96, 172]. We have also developed peptide microarray methods to profile protein kinase activities [31]. Peptide substrates were first immobilized on a glass slide through a long hydrophilic linker. After incubation with a protein kinase and [γ33P]-ATP, the phosphorylated peptides were detected by autoradiography or phosphorimager. This functional approach can be used to determine the substrate-specificity of a specific protein kinase, or to profile protein kinase activities in a complex biological sample such as cell extract or serum. Lizcano et al. studied the molecular basis for the substrate specificity of a human protein kinase, Nek6, using peptide microarrays containing more than 1000 different peptides [95]. They observed that protein kinase Nek6 required presence of leucine at the third position on the N-terminal side of the phosphorylation site. Recently, Houseman et al. and others reported the development of a peptide chip that could be phosphorylated by c-src protein kinase, and the level of phosphorylation could be determined by surface plasmon resonance, fluorescence and phosphorimaging [53, 161].

Like phosphorylation and dephosphorylation processes for signaling cascades, glycosylation of extracellular proteins and lipids are critical for the recognition of ligands and cell-cell interactions [48, 92, 167]. Houseman developed monosaccharide arrays and demonstrated that N-acetylglucosamine could be glycosylated by β-1,4-galactosyltransferase in the presence of the donor substrate UDPgalactose [34, 55].

Peptide substrates can also be used to profile protease activities. Salisbury et al. described a protease substrate microarray in which the carboxyl end of the peptide substrates was conjugated to 7-amino-4-carbamoylmethyl coumarin, a fluorogenic compound [143]. The conjugate was non-fluorescent when the electron-donating group on the coumarin was

attached to the peptide. Upon proteolysis, the peptide was released and the microarray spot fluoresced. In principle, substrate microarrays consisting of peptides or small molecules can be used as a valuable tool to profile many other enzymes. For example, Zhu et al. demonstrated that the small molecule microarrays could be used to detect enzyme activities of epoxide hydrolases and phosphatases [185].

8.6.3. Cell-Binding Studies

Protein or peptide microarrays can be used to profile the surface receptors or to study the biological function of a live cell. Belov et al. immobilized a series of cell surface marker specific antibodies to form an antibody microarrays, which was then used to profile cells present in the peripheral blood [10]. We have used OBOC combinatorial library methods to identify peptide ligands that bind the surface of intact cells [129]. We plan to develop these cell surface binding peptides into targeting agents for cancer [3]. Peptide microarrays can potentially be used as a diagnostic tool to profile patient cancer cells, allowing the physician can tailor an appropriate peptide cocktail for targeted therapy [3, 31, 83]. To test this concept, we immobilized 44 different cell-binding peptides onto polystyrene slides, and used intact Jurkat human T-lymphoma cells to probe the peptide microarray. The bound cells were stained with crystal violet. This micro cell adhesion assay enables us to identify those ligands that bind to live cancer cells [3]. Furthermore, the cell-binding assay, when used in conjunction with appropriate fluorescence labeled antibodies and confocal microscopy, will enable us to detect cell signaling or morphological changes of cells at spots where the cell attachment occurs [31].

8.6.4. Drug Discovery and Cell Signaling

The various approaches to prepare chemical libraries have been discussed in detail earlier in this review. Often, a combination of combinatorial techniques, in conjunction with standard medicinal chemistry, biophysical methods and molecular modeling methods are needed to develop a drug. Chemical libraries can also be prepared in a microarray format. Such libraries can be used for target validation and drug screening [16, 56, 159]. Although the number of chemical compounds one can generate in a chemical microarray format is limited (e.g., 5–10,000 compounds per slide as compared to 100,000–1,000,000 compounds per OBOC chemical library), replicates of chemical microarrays can be prepared and probed with a number of different target proteins. Schreiber et al. prepared OBOC macrobead chemical libraries, released the compound from each bead into micro wells and then printed chemical microarrays [15, 22]. In order to prepare enough compounds for a large number of microarray replicates, we used encoded bead-aggregates to prepare a “oneaggregate one-compound library” and released the compound from each aggregate into a 96-well plate, ligated the compound to agarose, and then printed the chemical microarray [102]. Such microarrays can then be screened with any of the detection methods discussed above.

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Elucidating these pathways requires methods to quantitate the activities of multiple proteins in a rapid and accurate manner. Multiplex antibody microarrays have been used to study the receptor tyrosine kinase signaling cascade in crude cell lysates [117]. Similarly,

chemical microarrays have been used to identify the small molecule ligands that can affect specific signaling proteins or pathways. For example, Schreiber and his co-workers reported the use of high-density small molecule arrays of 1, 3 dioxane small molecule library to identify the chemical ligands for the Ure2p transcriptional repressor in yeast [75]. The identified ligand, Uretupamine, was capable of modulating Ure2p signaling function inside the yeast cells. They also identified the small molecules that can interact directly to a signaling protein calmodulin and a yeast transcriptional factor Hap3p and demonstrated that the haptamide A inhibited the Hap3p functions in a dose dependent manner [70]. Housman et al. reported the use of peptide microarrays as a screening tool to simultaneously evaluate different substrates and inhibitors for c-src protein tyrosine kinase [54].

8.6.5. Diagnostic Studies

Diagnostic tests for determining serum antibody titers to a number of autoantigens, infectious agents, or other exogenous molecules have been used in clinical medicine for many years. Often these tests are performed in the clinical laboratory, one at a time, and require a large quantity of serum and reagents. In principle, all these tests could be miniaturized by immobilizing the antigens (proteins or peptides) in a microarray format. Similarly, antibodies specific to cytokines or other biological molecules can be immobilized on chips, which can be used either as diagnostic tools to evaluate serum levels of these biological molecules in patients or as research tools in proteomics [17, 156]. It is conceivable that within a decade, biochips will be available for clinical diagnosis, where hundreds to thousands of blood tests can be performed simultaneously and economically on each patient using only a minute amount of blood and analytes. Recent studies also suggest that peptide or small molecule microarrays are useful in the discovery of biomarkers for various diseases, such as autoimmune diseases and cancers [39, 130, 157]. Autoantibody profiles and IgE reactivity profiles have been created by arraying hundreds of autoantigens, including peptides, proteins, and other biomolecules and probing with normal and patients blood samples [51, 139]. Wang and co-workers used carbohydrate-based microarrays to analyze the different types of anticarbohydrate antibodies in human and mammalian sera [164, 165]. Interestingly, many of the carbohydrates that react with the sera are normally present in pathogenic microbes, suggesting that the individuals may have acquired these antibodies during a microbial infection. Very recently Amano et al. prepared peptide antigens decorated with various xenobiotics to evaluate their reactivities to sera derived from patients with primary biliary cirrhosis [5].

8.6.6. Non-Biological Applications

The non-biological applications of chemical microarrays, similar to that of combinatorial chemistry, have been lacking behind biological applications. There have been only a few reports on applying chemical microarrays to non-biological systems. Rakow et al. reported the development of a low-density chemical microarray of a limited number of compounds that can detect selected organic molecules [135]. In this method, a library of vapor-sensing metalloporphyrins dyes were immobilized on solid support. Visual identification of color change was easily achieved while a ligand was bound to the metalloporphyrins dyes. Using this method, a wide range of ligating vapor can be

detected even weakly ligating vapor such as halocarbons and ketones. This type of sensing array is of practical importance for general-purpose vapor dosimeters and analyte-specific detectors.

8.7. FUTURE DIRECTIONS

Since the early 1990s, the field of combinatorial chemistry has progressed rapidly and it has now become an indispensable tool for basic research and drug discovery. Microarrays, initially started as a form of a combinatorial peptide library, has evolved to DNA microarrays, protein microarrays, small molecule microarrays, and microarrays of many other biomolecules. These methods enable one to examine thousands of molecular interactions simultaneously. As a result, biological systems can be studied globally and efficiently. However, to fully exploit the potential of microarrays, more efficient and reproducible methods for immobilizing a uniform amount of chemical compounds or biomolecules onto solid surfaces need to be developed. Many effective detection systems for microarrays have already been described. The next challenge will be to develop mass spectroscopic methods, so that bound biomolecules to each microarray spot can be efficiently identified and quantified. DNA microarrays have already made a great impact in the field of genomics, and have begun to provide prognostic information for cancer patients. Protein, peptide, carbohydrate, and small molecule microarrays will continue to play an increasingly important role in the fields of proteomics, diagnostics, and drug development. We anticipate that in a decade, blood tests will no longer be performed one at a time. Instead, biochips that can perform hundreds to thousands of blood tests for each patient will become commonplace in modern medicine. As more proteins are cloned and expressed, proteome chips containing thousands of proteins will become available for probing protein-protein interactions, signaling pathways, and drug target identification. Human sub-proteome arrays, such as tissue specific and disease-specific protein collections, will enable researchers to rapidly characterize disease pathways for identification and validation of drug targets and biomarkers, as well as drug lead identification and global profiling of drug-protein interactions. Proteome microarrays derived from pathogens is expected to contribute greatly to the development of anti-infectious agents. Even though only a limited number of laboratories are currently working on small molecule microarrays, we expect research activities on this area will grow rapidly in the next few years. When probed with cell extracts obtained from normal and diseased tissues, these small molecule microarrays have the potential of generating drug leads, imaging agents, and drug targets at the same time. Furthermore, such microarrays may enable the researcher to isolate and identify protein complexes that are very important in cell signaling.

Combinatorial chemistry and chemical microarray techniques have already proven to be invaluable tools for biomedical research. Other areas that have and will continue to benefit from these new techniques are material science and sensor development. We anticipate that sensor chips based on chemical microarrays that can detect large numbers of environmental chemicals or biologicals will be developed. Material scientists will be developing methods to combinatorially generate large arrays of new material for rapid analysis. It is expected new materials will be discovered through this high-throughput approach.