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What do you do when you’re stuck with only a few cancer cells for analysis? “There is a lot of good work in the proteomic profiling of large numbers of cells in tissue samples,” explains Liang Li at the University of Alberta (Canada). “But we’re now facing analysis issues when we are confronted with small numbers of cells.” In a recent AC paper (DOI 10.1021/ac9023002), Li and colleagues address this problem with a new MS shotgun proteomic approach that can pick out 500⫺5000 cells from a sample for proteomic profiling. Take, for instance, circulating tumor cells (CTCs) that move through the bloodstream of cancer patients. CTCs have been linked to metastasis, but analyzing them has been a challenge because their numbers are so few. Culturing CTCs to generate large enough sample sizes for conventional MS proteomic approaches is not an option because the cells’ properties change when cultured. The investigators developed a multistep sample preparation procedure. The method looks simple on paperOlyse the cells with surfactant, precipitate proteins, and wash the protein pelletObut Li says it took a lot of effort to develop. The investigators had to test many lysis buffers and analyze their removal. Also, because they were starting off with limited sample sizes, Li and colleagues had to take great pains to minimize the loss of protein during the procedure. First, the researchers lysed the cells with the commercially-available NP-40 surfactant. “We couldn’t use techniques such as the French Press commonly used in dealing with larger numbers of cells. We had to use a chemical method that didn’t interfere with the downstream sample preparation and MS analysis,” explains Li. Li and colleagues had to find a way to remove the NP-40 to prevent interference with the MS analysis. “We precipitated all the protein out and used
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ANALYTICAL CHEMISTRY /
APRIL 1, 2010
LIANG LI
An MS method for proteomic analysis of small samples
Analysis of a small number of cancer cells includes collection of cells by flow cytometry, lysis, precipitation, washes, and an LC/MS run.
cold acetone to wash the pellet,” says Li. “The surfactant can be washed away. Any residual surfactant in the sample doesn’t interfere with our analysis.” Once they obtained their protein pellet, the investigators digested the proteins and carried out a nanoflow-LC/ Q-TOF analysis. They found they could generate proteomic profiles of ⬃150⫺650 proteins from 500, 1000, 2500, and 5000 cells. Karen Jonscher at the University of Colorado says the sample preparation procedure appears to be a fairly simple, robust, and reproducible method for analyzing very low cell numbers in blood. Given that CTCs can be indicators of potential metastases, “a noninvasive way to determine whether tumor cells are metastasizing or if a treatment appears to be reducing the number of CTCs is needed,” she explains.
The investigators demonstrated that their method could successfully analyze a small number of breast cancer cells spiked into a healthy human blood sample. They first added antibodies against the cancer cells to the spiked sample and collected the cells of interest by flow cytometry. Li and colleagues then applied their new method and showed they could generate proteomic profiles of the cancer cells. Li says the ultimate goal is to differentiate between cancer cells. “You can isolate cancer cells, but what type of cancer cells are you dealing with? Do they come from the lung, breast, or some other organ?” he says. “That’s the next challengeOto determine the specificity of the proteome profile that you generate from a small number of cells. The question is, can a small number of proteins be specific enough to pinpoint which type of cancer cells you’re dealing with?” Another important application, says K. W. Michael Siu of York University (Canada), is in laser capture microdissection, a technique in which researchers dissect out small numbers of cells. “If the methodology is done correctly, the cell population is more homogeneous because you can do a selection process,” he explains. With Li and colleagues’ method, “one could actually now do an analysis of selected cells effectively.” Li hopes that the work provides impetus to look into the analytical challenge of small cell numbers. “In the biology and medical fields, there is quite a bit of excitement about analyzing CTCs, but yet there are very few analytical tools available,” he says. “There is a lot of debate and argument over the significance of CTCs in metastasis. If we can get better chemical profiles of those CTCs and compare them to the real tumor cells in tissue, then perhaps we can get better links between CTCs and the tissue tumor cells.” —Rajendrani Mukhopadhyay
10.1021/AC100130P 2010 AMERICAN CHEMICAL SOCIETY
Published on Web 02/02/2010
