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Molecular & Cellular Proteomics 5:2392-2411, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Expanding the Subproteome of the Inner Mitochondria Using Protein Separation Technologies

One- and Two-dimensional Liquid Chromatography and Two-dimensional Gel Electrophoresis^*,S

Todd McDonald^,,¶, Simon Sheng^,¶, Brian Stanley^,,¶, Dawn Chen^||,**, Young Ko, Robert N. Cole^||,**, Peter Pedersen^|| and Jennifer E. Van Eyk^,,||,,

From the Departments of Medicine, ^|| Biological Chemistry, and Biomedical Engineering and ^** The Technical Implementation and Coordination Core of The Johns Hopkins NHLBI Proteomics Center, The Johns Hopkins University, Baltimore, Maryland 21224 and Department of Physiology, Queen’s University, Kingston, Ontario K7L 3N6, Canada

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab^TM PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the -subunit of F₁F₀ ATP synthase that differed due to a change in pI or hydrophobicity.

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