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 mitogenactivated 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.
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 g/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (ϳ22,500 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. Molecular & Cellular Proteomics 5:1658 -1666, 2006.The understanding of complex cellular networks necessitates tools that are amenable to the analysis of different parameters in a highly parallel manner (1). Although in the last years DNA microarrays were the technology of choice to monitor the abundance of several thousands of mRNA transcripts at a time, such studies provide us with little information on the proteins that are encoded by these transcripts (2, 3). However, because proteins rather than DNA carry out cellular functions, there is large interest to analyze proteins and their entirety, the proteome, in a manner comparable to DNA microarrays. One technology that is envisaged to meet the demands of high throughput protein interaction and modification screening is protein microarray technology (4 -8).Protein microarrays have been applied in different areas of application, such as the analysis of protein-protein interactions (9 -11), the identification of substrates for protein kinases (12-14), or the elucidation of potential diagnostic markers in bacterial or autoimmune diseases (15-18). All of them share the bas...
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. IntroductionThe global concept of array technology is the simultaneous analysis of thousands of molecules for a specific property under investigation. To this end, protein arrays were initially introduced to screen cDNA libraries for clones expressing recombinant proteins in Escherichia coli [1]. For this purpose, thousands of different expression clones were arrayed as bacteria on large protein binding membranes and -after induction and cell lysis -the presence of recombinant proteins on the array was correlated to individual clones. Subsequently, miniaturization has led to protein microarrays that are typically constructed by spotting protein samples onto microscope slides.Current protein microarrays come in a variety of formats. These include 'standard' protein microarrays (PMAs), which consist of purified recombinant proteins; antibody microarrays (AMAs); and reverse protein microarrays (RPMAs) generated from whole or fractionated cell lysates, as depicted in Figure 1a. Although the applications of PMAs can differ widely, the same general concept to detect interaction partners is applied in all. Putative binding partners are incubated with the arrayed proteins and binding is detected by using a label, either covalently bound to the putative interaction partner (Figure 1b) or a secondary antibody, or by novel label-free methods detailed below.In addition, the principle of delineating array results is the same for all PMA types; the signals -or labeled array spots -correlate the interaction to a known spot content according to the position on the array. Here, we discuss applications, technological advancements and detection systems developed in the past two years. Moreover, efforts towards standardization of protein microarray experimentation are reviewed. Protein microarraysThe earliest application of PMAs (Figure 1, left presenting a non-redundant set of approximately 1700 denatured Arabidopsis thaliana proteins were addressed with different mitogen activated protein (MAP) kinases [7 ]. Besides known and suspected targets, novel unpredicted kinase substrates such as transcription factors, histones, kinases and ribosomal proteins were identified.A recent example of protein-protein interaction screening was demonstrated by Kawahasi and colleagues using three selected pairs of model proteins known to interact [8 ]. All proteins were synthesized using a wheat-germbased cell-free protein translation system shown to be suitable for high-throughput protein exp...
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.
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