Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.
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.
Zischka
Hans
H
GSF Research Centre for Environment and Health, Institute of Human Genetics, Oberschleissheim, Germany.
Weber
Gerhard
G
Weber
Peter J A
PJ
Posch
Anton
A
Braun
Ralf J
RJ
Bühringer
Dietmute
D
Schneider
Ulrich
U
Nissum
Mikkel
M
Meitinger
Thomas
T
Ueffing
Marius
M
Eckerskorn
Christoph
C
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*
D024101:Mitochondrial Proteins; D020543:Proteome