An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using
metrizamide gradients to remove cytosolic and other subcellular contaminant
proteins; (b) separation of
mitochondrial protein complexes using
sucrose density gradients after solubilization with
n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the
sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target
spotting and automated spectral acquisition; and (e)
protein identification from mixtures of tryptic
peptides by high-precision
peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential
mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These
proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane
proteins such as the
adenine nucleotide translocase that are not recovered in 2D
gels. The technique described here should also be useful for the identification of new
protein-
protein associations as exemplified by the validation of a recently discovered complex that involves
proteins belonging to the
prohibitin family.