GreenPhos, a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants. In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes. This is largely due to the need to improve protein extraction efficiency from plant cells, which have a dense cell wall, and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis. To this end, we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis, rice, tomato, and Chlamydomonas reinhardtii, enabling in-depth identification with high quantitative reproducibility of about 11 000 phosphosites, the greatest depth achieved so far with single liquid chromatography-mass spectrometry (LC–MS) runs operated in a data-dependent acquisition (DDA) mode. The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time- and cost-effectiveness. The method, named GreenPhos, combined with single-shot LC–MS, enabled in-depth quantitative identification of Arabidopsis phosphoproteins, including differentially phosphorylated spliceosomal proteins, at multiple time points during salt stress and a number of kinase substrate motifs. GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides, which, if samples are further fractionated and analyzed by multiple LC–MS runs, could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.