title: Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling.
abstract: Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.
authors.affiliation: From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg;
authors.forename: Catrin S
authors.initials: CS
authors.lastname: Müller
authors.affiliation: From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg;
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Bildl
authors.affiliation: From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg;
authors.forename: Alexander
authors.initials: A
authors.lastname: Haupt
authors.affiliation: §Institute for Biochemistry and Molecular Biology, Stefan-Meier-Strasse 17, 79104 Freiburg;
authors.forename: Lars
authors.initials: L
authors.lastname: Ellenrieder
authors.affiliation: §Institute for Biochemistry and Molecular Biology, Stefan-Meier-Strasse 17, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg;
authors.forename: Thomas
authors.initials: T
authors.lastname: Becker
authors.affiliation: §Institute for Biochemistry and Molecular Biology, Stefan-Meier-Strasse 17, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg;
authors.forename: Carola
authors.initials: C
authors.lastname: Hunte
authors.affiliation: From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg;
authors.forename: Bernd
authors.initials: B
authors.lastname: Fakler
authors.affiliation: From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg; ‖Logopharm GmbH, Schlossstrasse 14, 79232 March-Buchheim, Germany uwe.schulte@physiologie.uni-freiburg.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Schulte
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004579:Electron Transport / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D024101:Mitochondrial Proteins