Mapping Plant Phosphoproteome with Improved Tandem MOAC and Label-Free Quantification.

Protein phosphorylation represents a fundamental and evolutionarily conserved type of posttranslational modification that regulates protein functions and cellular signal transmission. Due to the dynamic nature of protein phosphorylation processes in plant cells, large-scale studies of phosphoproteins face several challenges such as low stoichiometry in the modified peptides in a proteome, as well as heterogeneity of the phosphopeptides of a given protein. Here we describe an updated tandem MOAC combined phosphoprotein and phosphopeptide enrichment strategy, a scalable phosphoproteomics approach that allows identification of thousands of phophopeptides in plant materials in one LC-MS analysis. We implemented modifications to several steps of the original tandem MOAC procedure to increase the identification and quantification of phosphopeptides in a sample beginning with less amount of tissue and a smaller amount of extracted protein.