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Drawing a better map
CAMBRIDGE, Mass.—Cell biology has been a sticking point in pharma, biotech and other aspects of life sciences, because to get a true picture of what's happening inside live cells, researchers have to know the locations of thousands of proteins and other molecules—a complex landscape for which there is a mostly incomplete map. However, steps are being made to fill in the overwhelming blank space on that map, with one of the latest breakthroughs being the development of a technique by chemists at the Massachusetts Institute of Technology (MIT) that can tag all of the proteins in a particular region of a cell, allowing them to more accurately map those proteins.
The work has been carried out with the help of researchers at Harvard Medical School and the Broad Institute of MIT and Harvard, and the new method coming out of that work combines the strengths of two existing techniques: microscopic imaging and mass spectrometry.
The work started two or three years ago, recalls Dr. Alice Y. Ting, the Ellen Swallow Richards Associate Professor of Chemistry at MIT.
"I had been thinking about methods for analyzing endogenous, rather than recombinant, proteins in living cells," she says. "A major limitation of live-cell microscopy methods is that they almost always visualize recombinant proteins, which come with overexpression and tag-based artifacts. Mass spectrometry is one of the most powerful tools for studying endogenous proteins, and I wanted to find a way to apply it to living cells."
She says there are other subcellular regions that she and her colleagues are very interested in mapping using this new proteomic method, because "the biology there is very rich," and she sees great value in this new method, which she says bridges the strengths of microscopy on the one hand and mass spectrometry on the other hand.
"Two major methods exist for studying proteins inside the cell: mass spectrometry and imaging of fluorescently tagged proteins. These methods complement each other in terms of strengths and weaknesses," explains Dr. Peng Zou of MIT, who works with Ting at MIT. "On the one hand, mass spectrometry is capable of analyzing thousands of proteins in parallel but does not provide any information about the spatial arrangement of these proteins in the cells; on the other hand, imaging reveals the detailed localization information of proteins, but it analyzes only a handful of proteins—less than 10—at a time. We sought to combine the strengths of these two methods, so as to uncover the protein contents at specific locations inside the cell. This requires tagging proteins with a promiscuous labeling enzyme. We went ahead to engineer a peroxidase to achieve this goal."
Looking at next steps, Zou says, now that they have demonstrated this method in the mitochondrial matrix, they are interested in extending its applications to such cellular compartments as the endoplasmic reticulum and the mitochondrial intermembrane space. These experiments are in progress, but Zou stresses that as promising as this new technique is, it is still very much in its infancy. To obtain a true proteomic map of the cell would requires 100-percent coverage of the underlying proteome, without bias toward amino acid residues and complete control of labeling radius, which are among the goals Ting, Zou and the other researchers are working toward.
The mitochondrial matrix that is the topic of the paper published by Ting's team in the Jan. 31 online edition of Science can be purified by density centrifugation, although the quality of the preparation is poor, Ting notes.
"I think where this new method will have the greatest impact is in studying the proteomes of cellular regions that are impossible to purify—for example, non-membrane bounded cellular compartments like the synaptic cleft," she says, adding, "There are potential applications for drug discovery and understanding disease mechanisms. For example, one could envision using the mitochondrial mapping scheme we report in the paper to analyze patient-derived cells. What are the proteome-wide differences between cells from healthy people versus people with mitochondrial disease? How do these proteomes change in response to therapeutic intervention? Our method uses very little material and is simple to implement and therefore may be a practical tool for studying disease."
In terms of additional applications and breakthroughs, Zou notes that so far, their method has revealed that the subcellular location of a heme-biosynthesis enzyme has been incorrectly assigned in the past.
"This brought up the question of the presence of an unexpected transporter that might have escaped notice in the previous model. This information is potentially useful for understanding genetic diseases related to heme," Zou says. "Although our method does not impact the pharmaceutical industry directly, it helps reveal the protein composition at specific cellular locations. Such information could be useful for diagnosis."