title: Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1.
abstract: We describe in detail the usage of leucine metabolic labelling in yeast in order to monitor quantitative proteome alterations, e.g. upon removal of a protease. Since laboratory yeast strains are typically leucine auxotroph, metabolic labelling with trideuterated leucine (d3-leucine) is a straightforward, cost-effective, and ubiquitously applicable strategy for quantitative proteomic studies, similar to the widely used arginine/lysine metabolic labelling method for mammalian cells. We showcase the usage of advanced peptide quantification using the FeatureFinderMultiplex algorithm (part of the OpenMS software package) for robust and reliable quantification. Furthermore, we present an OpenMS bioinformatics data analysis workflow that combines accurate quantification with high proteome coverage. In order to enable visualization, peptide-mapping, and sharing of quantitative proteomic data, especially for membrane-spanning and cell-surface proteins, we further developed the web-application Proteator (http://proteator.appspot.com). Due to its simplicity and robustness, we expect metabolic leucine labelling in yeast to be of great interest to the research community. As an exemplary application, we show the identification of the copper transporter Ctr1 as a putative substrate of the ER-intramembrane protease Ypf1 by yeast membrane proteomics using d3-leucine isotopic labelling.
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Nilse
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
authors.forename: Dönem
authors.initials: D
authors.lastname: Avci
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Heisterkamp
authors.affiliation: Freie Universität Berlin, Department of Informatik, Takustr. 9, 14195 Berlin, Germany; Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelsee 310, 12587 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Serang
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. Electronic address: m.lemberg@zmbh.uni-heidelberg.de.
authors.forename: Marius K
authors.initials: MK
authors.lastname: Lemberg
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany. Electronic address: oliver.schilling@mol-med.uni-freiburg.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D019295:Computational Biology / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D007930:Leucine / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015003:Yeasts / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; D010447:Peptide Hydrolases; D007930:Leucine
keywords: Feature detection; Isotope labelling; Proteolysis