Researchers from North Carolina State University have developed a new technique to identify the proteins secreted by a cell. The new approach should help researchers collect precise data on cell biology, which is critical in fields ranging from zoology to cancer research.
The work is important because cells communicate by secreting proteins. Some of the proteins act on the cell itself, telling it to grow or multiply, for example. But the proteins can also interact with other cells, influencing them to perform any biological function.
Traditionally, scientists who wanted to identify these proteins cultured cells and then used mass spectrometry to determine which proteins appeared in the medium the cell was grown on. This has drawbacks, because the proteins of interest are fairly rare compared to the proteins that are already in the medium -- which are used to grow and support the cells in the first place. Further, any attempts to culture the cells without these background, supporting proteins affects cell behavior -- skewing the sample.
The new approach takes advantage of the fact that each cell "packages" its proteins in its "secretory pathway." Each cell synthesizes the protein and passes it through this pathway, essentially placing it in a bag-like membrane before it is passed out of the cell.
In their new technique, researchers take a sample of cells and isolate the secretory pathway organelles, which contain the proteins. The researchers then use mass spectrometry to analyze the contents of the organelles, in order to see which proteins were being secreted by the cell. Using this approach, the researchers were able to identify proteins that are secreted by human embryonic stem cells.
"This gives us a snapshot of exactly what a cell was secreting at that point in time," says Dr. Balaji Rao, an assistant professor of chemical and biomolecular engineering at NC State and co-author of a paper describing the work.
This new method eliminates the problems related to the proteins found in cell culture media. But it also allows researchers to track changes in the proteins released by a cell in response to a stimulus, such as exposure to a chemical. This can be done by taking samples at various points in time after cells have been exposed to the stimulus.
And, in principle, this technique would also allow researchers to identify which proteins any specific type of cell is secreting when in a mixed population of cells.
"As long as you can separate the cells you are interested in, this should be possible," says Rao. "And that is important, because most tissues are made up of heterogeneous populations of cells -- and communication between those cells is biologically significant."
The paper, "Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonic fibroblasts and human embryonic stem cells," was published online Sept. 15 in the journal Molecular & Cellular Proteomics. Lead author of the paper is Prasenjit Sarkar, a Ph.D. student at NC State. Co-authors are Rao; NC State Ph.D. student Shan Randall; and Dr. David Muddiman, a professor of chemistry at NC State. The research was funded by the National Science Foundation.
- P. Sarkar, S. M. Randall, D. C. Muddiman, B. M. Rao. Targeted proteomics of the secretory pathway reveals the secretome of mouse embryonicfibroblasts and human embryonic stem cells.. Molecular & Cellular Proteomics, 2012; DOI: 10.1074/mcp.M112.020503
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