title: Identification of MAPK Substrates Using Quantitative Phosphoproteomics.
abstract: Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL, 32610, USA.|Genetics Institute, University of Florida, Gainesville, FL, 32610, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL, 32610, USA.|Department of Chemical Engineering, University of Florida, Gainesville, FL, 32610, USA.
authors.forename: Jacqueline D
authors.initials: JD
authors.lastname: Schneider
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL, 32610, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhu
authors.affiliation: Department of Biology, University of Florida, Gainesville, FL, 32610, USA. schen@ufl.edu.|Genetics Institute, University of Florida, Gainesville, FL, 32610, USA. schen@ufl.edu.|Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, 32610, USA. schen@ufl.edu.|Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, 32610, USA. schen@ufl.edu.
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
mesh_terms: D005818:Genetic Engineering; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis*; D010940:Plant Proteins / Q000032:analysis; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010750:Phosphoproteins; D010940:Plant Proteins; D020928:Mitogen-Activated Protein Kinases
keywords: MAPK; MAPK substrate; Phosphoproteomics; Signal transduction; TMT