title: Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.
abstract: The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science , Stanford, California 94305, United States.|Institute of Biosciences and Bioresources (CNR-IBBR), National Research Council of Italy , Palermo 90129, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: de Michele
authors.affiliation: Department of Botany, University of British Columbia , Vancouver, BC V6T 1Z4, Canada.|Max Planck Institute for Molecular Plant Physiology, Potsdam 14476, Germany.
authors.forename: Heather E
authors.initials: HE
authors.lastname: McFarlane
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|Department of Plant and Environmental Sciences, University of Copenhagen , Copenhagen C-1871, Denmark.
authors.forename: Harriet T
authors.initials: HT
authors.lastname: Parsons
authors.affiliation: Department of Botany, University of British Columbia , Vancouver, BC V6T 1Z4, Canada.
authors.forename: Miranda J
authors.initials: MJ
authors.lastname: Meents
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Jeemeng
authors.initials: J
authors.lastname: Lao
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Susana M
authors.initials: SM
authors.lastname: González Fernández-Niño
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science , Stanford, California 94305, United States.
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
authors.affiliation: Department of Botany, University of British Columbia , Vancouver, BC V6T 1Z4, Canada.
authors.forename: A Lacey
authors.initials: AL
authors.lastname: Samuels
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|ARC Centre of Excellence in Plant Cell Walls, School of Botany, The University of Melbourne , Melbourne, Victoria 3010, Australia.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020543:Proteome / Q000302:isolation & purification* / Q000378:metabolism; D036226:Seedlings / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D020543:Proteome
keywords: Arabidopsis; free-flow electrophoresis; plasma membrane