Recent advances of protein microarrays.

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.

Immunization with hybrid proteins containing the membrane proximal external region of HIV-1.

The transmembrane envelope (TM) protein gp41 of HIV-1 is an attractive target when designing a vaccine to induce neutralizing antibodies. A few broadly neutralizing antibodies (2F5, 4E10, and 10E8) that target conserved epitopes in the membrane proximal external region (MPER) of gp41 have been isolated from infected individuals. However, attempts to induce such antibodies by immunizations with gp41 and Env derivatives containing the MPER were successful only to some extent. In contrast, immunizations with the ectodomain of the TM protein p15E of different gamma retroviruses resulted in the induction of neutralizing antibodies. These sera recognized epitopes located in the MPER and in the fusion peptide proximal region (FPPR) of p15E. Based on these results, both regions of p15E were substituted with the corresponding sequences derived from gp41 of HIV-1. Thus, four different hybrid antigens were produced. One of the inserted sequences contained the epitopes of 2F5 and 4E10 in the MPER; the other corresponded to the FPPR. Vaccination of rats, guinea pigs, and a goat induced binding antibodies directed against the FPPR of gp41 and the 2F5 epitope (ELDKWA) located in the MPER. Despite the exact recognition of the 2F5 epitope, no or very weak neutralization of HIV-1NL4-3 by the immune sera was demonstrated. Nonetheless, using the strategy of hybrid proteins, antibodies targeting the desired epitope were successfully induced.

An integrative approach for analyzing the interplay of genetic and epigenetic changes in tumors.

The accumulation of chromosomal aberrations is a characteristic feature of tumor development. However, an understanding of tumorigenesis that assumes that changes in DNA copy number always cause equivalent changes in the corresponding RNA and protein levels is an oversimplification and completely ignores the individual genetic and epigenetic context in which an aberration has to be evaluated. We present a brief introduction to various techniques dedicated to the genome-wide analysis of genetic and epigenetic changes, and illustrate how complementary information derived from these various DNA array-based technologies can lead to a better understanding of the consequences of chromosomal aberrations.

Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein-protein interactions or protein-small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.

Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products.

Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 microg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product ( approximately 22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones.

Subnanoliter enzymatic assays on microarrays.

Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.

Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays.

Automation is the key approach for genomewide and proteomewide screening of function and interaction. Especially for proteomics, antibody microarrays are a useful tool for massive parallel profiling of complex samples. To meet the requirements of antibody microarrays and to obtain a great variety of antibodies, new technologies such as phage display have partly replaced the classical hybridoma method. While the selection process for phage-displayed antibody fragments itself has been automated, the bottleneck was shifted further downstream to the identification of monoclonal binders obtained from the selections. Here, we present a new approach to reduce time, material, and waste to extend automation beyond the selection process by application of conventional microarray machinery. We were able to express recombinant antibody fragments in a single inoculation and expression step and subjected them without purification directly to an automated high-throughput screening procedure based on the multiple spotting technique (MIST). While obtaining comparable sensitivities to enzyme-linked immunosorbent assays, we minimized manual interaction steps and streamlined the technique to be accessible within the automated selection procedure.