High-throughput identification of plant protein kinase substrates

Proteomic approaches, such as protein microarray technology, play an important role in the study of complex biological systems. Their application in plant science has been strongly supported by the completion of genome sequence projects in the model plants Arabidopsis thaliana and rice. This chapter focuses on the identification of substrate phosphorylation by protein kinases using protein microarrays. Protein microarrays are widely used to profile antibodies and sera for their specificity, and/or to screen entire proteomes for new protein interactions. Here, we emphasise the feasibility of using protein microarrays in the discovery of potential protein kinase substrates in plants. Our group has identified potential substrates in two different plant species: first barley and later Arabidopsis. A signal quantification and threshold-based selection method was introduced during optimisation of protein microarray technology for substrate identification in order to better evaluate the microarray data, and to provide a preliminary list of candidate substrates for further investigation.