Spatial and Temporal Proteomic Analysis in the Mitochondria of…

Mass spectrometry and microscopy have always been considered two separate but complementary research techniques. Mass spectrometry has the ability to identify thousands of proteins through proteomic pipelines, but only in lysed organelles or cells. Microscopy, on the other hand, is capable of identifying the spatial and temporal location of proteins, but only a few at a time. Therefore, a combination of the two would provide a valuable tool capable of identifying numerous proteins in time and space across living cells.

The Ting group at the Department of Chemistry at the Massachusetts Institute of Technology (MIT) has been working on this very problem of combining mass spectrometry and microscopy. APEX (engineered ascorbate peroxidase) is a compound that was developed for electron microscopy and is catalyzed by the addition of peroxides and phenols to create a phenoxy radical that can then bind to the protein. The reaction is covalent and occurs at electron-rich amino acids, such as tyrosine, tryptophan, histidine, and cysteine. The radical formation is very short lived (less than 1 msec), and the labeling radius is very small (less than 20 nm).

The Ting group tested this chemistry by creating a fusion protein with a 24-amino-acid peptide that targets the mitochondria. The labeling of mitochondrial proteins was initiated through the addition of biotin-phenol and 1 mM H₂O₂. Cells were then monitored by the addition of neuravidin to stain and visualize the cells under fluorescence microscopy, while other cells were lysed and the proteins were purified using streptavidin beads. The proteins were eluted off the streptavidin, separated by SDS-PAGE, digested, and analyzed by mass spectrometry. Using stable isotope labeling, the Ting lab labeled experimental and control samples to distinguish between the biotinylated and the nonspecific binders. From their experiment, 495 proteins were identified. From those proteins, 31 were orphan proteins that were not considered to be located near to the mitochondria. Their localization was confirmed by imaging that showed either partial or complete localization. Another 240 proteins of unknown mitochondrial localization were identified as localized to the mitochondrial matrix. Also, six proteins that were previously assigned to the outer mitochondrial membrane were also identified as being localized to the mitochondrial matrix.

The Ting group used seven other APEX fusion proteins to test the approach with cytosolic proteins. Each of the seven fusion proteins gave a different subset of cytosolic proteins resulting from the small labeling radius.

APEX labeling or other types of fusion protein labeling systems similar to this are ideal for proteomics within a region of living cells as well as being able to capture an image of their localization. The chemistry can be specific to target different organelles, while the actual protein labeling is sufficiently general to be able to capture all the proteins within the targeted area. The applications for proteomics, disease proteomics, and systems biology is great and may help uncover new information about cellular proteomic organization.

References

1. Rhee, H.-W., et al. (2013) ‘Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging‘, Science, 339 (6125), (pp. 1328-1331)