The concept of microarrays was developed from an earlier concept termed ambient analyte immunoassay, first introduced by Roger Ekins in 1989. In the following decade, microarrays were first successfully realized as DNA or oligonucleotide microarrays, which allowed the quantification of the mRNA expression levels of thousands of genes in parallel. This technology has changed many aspects of biological research. Though extremely successful, the chemistry of DNA hybridization precludes its application for studying proteins, which are considered the major driving force in cells. Consistent with this view, mRNA profiles do not always correlate with protein expression as reported in many recent mass spectrometry studies [1–3]. Therefore, protein microarrays were developed as a high-throughput tool to overcome the limitations of DNA microarrays, and to provide a versatile platform for protein functional analyses [4–6].
At the beginning of the development of protein array technology, bacterial strains of a cDNA expression library were gridded and grown on nylon membranes, followed by lysis of the bacteria and immobilization of the total protein complement [7, 8]. However, these early attempts only had limited success, because 1) heterologous proteins (e.g., human proteins) were expressed in bacteria, yielding proteins that lacked critical eukaryotic posttranslational modifications; 2) denaturing conditions were used to lyse the bacterial host, resulting in improperly folded proteins; 3) proteins of interest were not purified away from thousands of unwanted bacterial proteins; and 4) the density of the array was low. Before long, other research groups began to report their efforts to fabricate high-density protein microarrays with purified proteins or antibodies [9–12]. In order to improve protein stability and preserve the native conformation of purified proteins, many research groups developed a variety of surface features to keep proteins hydrated during protein microarray fabrication. These efforts included reports on the 3D gel-pad chips , nanowell chips , and plasma membrane-coated chips , to name a few.
The real breakthrough was a 2001 report on the fabrication of a yeast proteome microarray by the Snyder group . In this study, approximately 5,800 full-length yeast ORFs were individually expressed in yeast and their protein products purified as N-terminal GST-fusion proteins. Then, each purified protein was robotically spotted on a single glass slide in duplicate at high-density to form the first “proteome” microarray, as it covered more than 75% of the yeast proteome. More recently, proteome microarrays have been fabricated from the proteomes of viruses, bacteria, plants, and humans [4, 16–21].
On the basis of their applications, protein microarrays can be divided into two classes: analytical and functional protein microarrays . Unlike antibody arrays (analytical microarrays), functional protein microarrays are made by spotting purified proteins on solid surfaces and are therefore useful for direct characterization of protein functions, such as protein binding properties, posttranslational modifications, enzyme-substrate relationships, and immune responses [5, 22]. More recently, a reverse-phase array was developed in which tissue or cell lysates, as opposed to antibodies, are used to construct the array .
Meanwhile, we and others have developed various types of biochemical assays that can be conducted using protein microarrays to characterize protein-binding properties, including protein-protein, -DNA, -RNA, and, -lipid interactions, and to identify substrates of various types of enzymes, such as protein kinases, acetyltransferases, and ubiquitin and SUMO E3 ligases via covalent reactions [9, 15, 24–31] (Table 1). These efforts clearly demonstrate the versatility and power of protein microarray technology as a systems biology and proteomics tool [6, 32]. In this review, we will summarize recent applications of protein microarrays in clinical proteomics, including biomarker identification, pathogen-host interactions, and cancer biology (Table 2).