首页|GreenPhos,a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility
GreenPhos,a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility
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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 phos-phoproteomes.This is largely due to the need to improve proteinextraction 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 purifica-tion 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 includingArabi-dopsis,rice,tomato,and Chlamydomonas reinhardtii,enabling in-depth identification with high quantita-tive 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 spec-trometry technology.
State Key Laboratory of Molecular Developmental Biology,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China
University of Chinese Academy of Sciences,Beijing 100049,China
Key Laboratory of Photobiology,Institute of Botany,Chinese Academy of Sciences,Beijing 100093,China
Ministry of Science and Technology of the People's Republic of ChinaMinistry of Science and Technology of the People's Republic of ChinaStrategic Priority Research Program of Chinese Academy of SciencesNational Key Research and Development Program of China
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