Reporter: Aviva Lev-Ari, PhD, RN
Between the maze-like inner membranes of the mitochondria, there’s a thick, sticky region called the matrix. This region serves an important role in the generation of the cell’s energy, but the proteins that actually make up this matrix have remained a mystery. But now, researchers at the Massachusetts Institute of Technology (MIT) have catalogued all the proteins in the mitochondrial matrix, identifying 31 proteins not previously associated with mitochondria. They did this by combining the strengths of two methods: microscopy and mass spectrometry.
Electron microscopy of human embryonic kidney cells expressing mito-APEX. Credit: Alice Ting, MIT
“This method is really a new paradigm for doing mass spec proteomics because we’re recording proteomics in living cells,” said Alice Ting, a chemist at MIT and author of a paper published online yesterday in Science that describes the technique (1).Microscopy and mass spectrometry are valuable for studying proteins, but each has its drawbacks. While microscopy can show where a protein is located within a cell, it can only do so for a small number of a cell’s roughly 20,000 proteins at once. Meanwhile, mass spectrometry can identify all the proteins within a cell, but destroys the cell membrane in the process of releasing the cell’s contents, resulting in a mixture of proteins from different cell regions and organelles.
To overcome these limitations, Ting’s group genetically engineered the mitochondrial matrix to express a newly designed peroxidase called APEX. When biotin-phenol was added to these cells, APEX stripped an electron and a proton from the biotin molecule, creating highly reactive biotin-phenoxyl radicals. These radicals quickly bound to nearby proteins to stabilize themselves, effectively tagging the proteins in the matrix.
The scientists then identified these tagged proteins with fluorescent imaging, dissolved the cell membrane, and isolated the proteins from the mitochondrial matrix. Using mass spectrometry, the team then identified 495 proteins in the mitochondrial matrix, 31 of which had not been previously linked to the mitochondrial region.
One of the biggest surprises was the discovery that the enzyme PPOX is in the matrix. PPOX helps synthesize heme, the pigment in red blood cells and a cofactor of the protein hemoglobin. Previously, biologists believed that PPOX was located within the space between the outer and inner membranes of the mitochondria, but Ting’s team found that it was actually within the matrix, which the team said is an example of how locally precise their biotin-tagging technique is.
Now, Ting and her team are looking at proteins in the mitochondrial intermembrane space. In addition, the researchers are tweaking their labeling system to map proteins in the cell membrane and to detect specific protein-protein interactions.
Reference
1. Rhee, H.-W., P. Zou, N. D. Udeshi, J. D. Martell, V. K. Mootha, S. A. Carr, and A. Y. Ting. 2013. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science (January).
SOURCE:
http://www.biotechniques.com/news/biotechniquesNews/biotechniques-339645.html#.UQ37hRxiB0w
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