title: A proteomic strategy for global analysis of plant protein complexes.
abstract: Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Uma K
authors.initials: UK
authors.lastname: Aryal
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Zachary
authors.initials: Z
authors.lastname: McBride
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Department of Computer Science, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kihara
authors.affiliation: Department of Statistics, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Jun
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Hall
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Department of Agronomy, Purdue University, West Lafayette, Indiana 47907 dszyman@purdue.edu.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Szymanski
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis / Q000145:classification; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D003600:Cytosol / Q000378:metabolism; D015151:Immunoblotting; D013058:Mass Spectrometry; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis* / Q000145:classification; D020543:Proteome / Q000032:analysis* / Q000145:classification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; D020543:Proteome