title: Approaches to Characterize Organelle, Compartment, or Structure Purity.
abstract: The function of subcellular structures is defined by their specific sets of proteins, making subcellular protein localization one of the most important topics in organelle research. To date, many organelle proteomics workflows involve the (partial) purification of the desired subcellular structure and the subsequent analysis of the proteome using tandem mass spectrometry (MS/MS). This chapter gives an overview of the methods that have been used to assay the purity and enrichment of subcellular structures, with an emphasis on quantitative proteomics using differently enriched subcellular fractions. We introduce large-scale-based criteria for assignment of proteins to subcellular structures and describe in detail the use of15N metabolic labeling in moss to characterize plastid and mitochondrial proteomes.
authors.affiliation: Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany.
authors.forename: Stefanie J
authors.initials: SJ
authors.lastname: Mueller
authors.affiliation: Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany.
authors.forename: Sebastian N W
authors.initials: SN
authors.lastname: Hoernstein
authors.affiliation: Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany. ralf.reski@biologie.uni-freiburg.de.|BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany. ralf.reski@biologie.uni-freiburg.de.|FRIAS Freiburg Institute for Advanced Studies, University of Freiburg, Freiburg, Germany. ralf.reski@biologie.uni-freiburg.de.|TIP Trinational Institute for Plant Research, University of Freiburg, Freiburg, Germany. ralf.reski@biologie.uni-freiburg.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Reski
mesh_terms: D019068:Bryopsida / Q000737:chemistry* / Q000648:ultrastructure; D002458:Cell Fractionation / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry / Q000648:ultrastructure; D009587:Nitrogen Isotopes / Q000378:metabolism; D015388:Organelles / Q000737:chemistry* / Q000648:ultrastructure; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018087:Plastids / Q000737:chemistry / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D013347:Subcellular Fractions / Q000737:chemistry / Q000648:ultrastructure; D053719:Tandem Mass Spectrometry
chemical_list: D009587:Nitrogen Isotopes; D010940:Plant Proteins
keywords: Compartment marker; Density gradient purification; Metabolic labeling; Mitochondria; Physcomitrella patens; Plastid; Quantitative proteomics