Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free‐flow electrophoresis

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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.