Plant Phosphopeptides Enrichment by Immobilized Metal Ion Affinity Chromatography

Protein phosphorylation plays important roles in the regulation of plant growth and development as well as adaption to changing environments. Large-scale identification of the phosphorylated proteins could provide both a global view of and specific targets involved in the mechanism underlying these processes. The progress of phosphoproteomic study for higher plants has lagged behind that of animals due to technical challenges, particularly the difficulty in solubilizing proteins from plant tissues with a rigid cell wall and the interference of the secondary metabolites, polysaccharides, and pigments throughout the whole processes of sample preparation and LC-MS analysis. Thus, it is critical to improve the efficiency of protein extraction and to remove the interfering metabolites before phosphopeptides enrichment. Here we describe a protocol for plant protein extraction and phosphopeptides enrichment by Fe³⁺-immobilized metal ion affinity chromatography (Fe³⁺-IMAC). Strong detergents such as SDS were used to extract proteins from plant tissues, and the secondary metabolites were removed by protein precipitation and washing of the pellets. The protein samples were digested and the resulting peptides were prefractionated. Phosphopeptides enriched from each fraction were combined before analysis with LC-MS.