title: Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.
abstract: The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.
authors.affiliation: GSF Research Centre for Environment and Health, Institute of Human Genetics, Oberschleissheim, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Zischka
authors.forename: Gerhard
authors.initials: G
authors.lastname: Weber
authors.forename: Peter J A
authors.initials: PJ
authors.lastname: Weber
authors.forename: Anton
authors.initials: A
authors.lastname: Posch
authors.forename: Ralf J
authors.initials: RJ
authors.lastname: Braun
authors.forename: Dietmute
authors.initials: D
authors.lastname: Bühringer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schneider
authors.forename: Mikkel
authors.initials: M
authors.lastname: Nissum
authors.forename: Thomas
authors.initials: T
authors.lastname: Meitinger
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.forename: Christoph
authors.initials: C
authors.lastname: Eckerskorn
mesh_terms: D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods; D005240:Feasibility Studies; D007091:Image Processing, Computer-Assisted; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D020543:Proteome