Protein phosphorylation is a key signaling mechanism during the plant biotic and abiotic stress response. Signaling cascades communicate between the cell surface, where the stress is perceived, and the nucleus, where a response can be enacted. Many of these signals involve the specific, transient phosphorylation of proteins by kinases, a signal which is usually amplified through cascades. The advent of high-throughput phosphoproteomics, pioneered mainly in yeast and mammalian cells, has made it possible to discover novel phosphorylation events rapidly and efficiently in a data-dependent manner and this has greatly enlarged our understanding of the plant's response to stress. This chapter describes a simple gel-free protocol for high-throughput phosphoproteomics, which is amenable to most labs engaged in plant stress research.
Keywords: IMAC; Phosphoproteomics; Signaling; TiO2.