Mild protease treatment as a small-scale biochemical method for mitochondria purification and proteomic mapping of cytoplasm-exposed mitochondrial proteins.
Because of its importance in basic biology and medicine, great efforts are being devoted to unraveling of the genuine mitochondrial proteome, which is the dynamic protein complement that the organelle uses to maintain its structure and functionality. Several proteomic investigations have now clearly shown that all the purification approaches we have at our disposal suffer from the problem of co-purification; therefore, it is very difficult to distinguish novel mitochondrial proteins from those that are just contaminants of the preparation. The question is further complicated by the fact that the mitochondrial proteome depends on the tissue source. Density gradient centrifugation is the most widespread purification method for obtaining highly pure mitochondrial fractions. The main disadvantage of these methods is the low yield of purified mitochondria that precludes their use in low-scale purifications. Here, we have treated small aliquots of crude mitochondria from mouse liver and from cultured hepatocytes (HEPA1-6) with trypsin under mild proteolysis conditions and have evaluated the suitability of this reaction as a small-scale purification approach. The protease removed several cytoplasmic and endoplasmic reticulum proteins, together with a fraction of mitochondrial proteins that we hypothesize to be associated with the cytosolic face of the outer mitochondrial membrane. The peculiar topology of these mitochondrial proteins could be indicative of their functional roles. Finally, our study represents an application of advanced mass spectrometry technology to the evaluation of biochemical approaches for the treatment of mitochondria.
Forner
Francesca
F
Max Planck Institute for Biochemistry, Department of Proteomics and Signal Transduction, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Arriaga
Edgar A
EA
Mann
Matthias
M
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D019295:Computational Biology; D003593:Cytoplasm / Q000378:metabolism; D005069:Evaluation Studies as Topic; D022781:Hepatocytes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D024101:Mitochondrial Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
D024101:Mitochondrial Proteins; D014357:Trypsin