Generation of plant protein microarrays and investigation of antigen-antibody interactions.

The application of proteomics methods, such as the protein microarray technology, in plant science has been strongly supported by the completion of genome sequencing projects of Arabidopsis thaliana and rice. In this chapter we describe a method to generate plant protein microarrays and to use them for characterizing monoclonal antibodies or polyclonal sera with regard to their specificity and cross-reactivity. The method starts with characterized E. coli cDNA expression clones encoding His-tagged plant proteins. After expression and purification of these recombinant proteins in high throughput, protein microarrays are generated utilizing a contact printer. For the detection of the recombinant proteins on the microarrays, an anti-RGS-His6 antibody is used. To characterize specific antibodies, the microarrays are incubated with the respective antibody solutions followed by fluorescently labeled secondary antibodies. Signal detection is performed by means of an arrayscanner system. Protein microarrays containing the whole proteome of a plant will represent the ideal format to test antibody specificity and cross-reactivity in the future.

Phosphorylation studies using plant protein microarrays.

Identifying protein kinase substrates is one major focus of protein kinase research and supports the elucidation of signal transduction pathways and their complex regulation. In this chapter we describe a protein microarray-based in vitro method, which permits a systematic screening of immobilized proteins for their phosphorylation by specific protein kinases. This high-throughput method allows the identification of potential kinase substrates. The method starts with plant protein microarrays containing hundreds of purified recombinant His-tagged proteins. The microarrays have to be incubated with the soluble and active kinase in the presence of radioactive [gamma33-P]ATP. Only small volumes of active kinase are needed for one microarray experiment. Radioactive signals are then detectable by phosphor imager or X-ray film. The identified potential substrates have to be verified by other in vitro or in vivo methods, as both the kinase and the substrate may not interact with each other under in vivo conditions. This screening method could be generally applicable for direct identification of candidate substrates of various protein kinases.

Identification of barley CK2alpha targets by using the protein microarray technology.

We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of [gamma-33P]ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin.

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.