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.
Keywords: mitochondria • outer membrane • small-scale • purification • proteolysis • mass spectrometry • orbitrap
• organellar proteomics