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Proteomics gets out of a fix
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A N A LY T I C A L C H E M I S T R Y / D E C E M B E R 1 , 2 0 0 5
lost some proteins because endogenous proteases may have been activated when we processed it, just as they become activated when you tear up lettuce and it becomes mushy,” Palmer-Toy says. He also analyzed a piece of spiral ligament from a cochlea that had been fixed and embedded 4 years earlier, identifying 125 proteins. “We were particularly pleased with the high sequence coverage that we observed for proteins of interest,” he notes, cautioning against putting too much stock in the total number of proteins “identified” in the various reports. “We were worried about misidentifying peptides modified during fixation, so we used very stringent identification criteria and estimated our misidentification frequency by using a reversed protein database,” he adds. Palmer-Toy says his group’s protocol is likely to work with a wide range of FFPE tissues. He explains, “We don’t see that there is going to be any additional challenge posed by other tissue types, considering that some of the materials we work with, including calcified tissues, are generally the most difficult to work with.” According to Palmer-Toy, the group’s next challenge will be to extract proteins from celloidinembedded tissues. At the University of Utah, Kojo Elenitoba-Johnson removes the paraffin from diced paraffin blocks with xylene (Lab. Invest. 2005, doi 10.1038/labinvest. 3700343). After hydrating the sample in graded ethanols, he washes it in a mixture of Tris HCl, Triton X-100, sodium deoxycholate, sodium chloride, and EDTA. The proteins are then digested with trypsin or glutamic C endopeptidase and analyzed by MS. Testing this protocol on a 3-year-old FFPE cell block of a human lymphoma cell line and using the criterion of at least 2 unique peptides, he identified 324 proteins, compared with 514 in a frozen SAUMIL MERCHANT, MEEI
Darryl Erik Palmer-Toy and colLike manuscripts in a vault, disease-releagues of the Harvard Partners Center lated proteins are locked away in the for Genetics and Genomics, Massachuvast collection of formalin-fixed, parafsetts General Hospital, and the Massafin-embedded (FFPE) tissues stored by chusetts Eye and Ear Infirmary (MEEI) the world’s pathologists. If only these have developed a nonproprietary method proteins could be liberated, scientists could delve into the remains of clinical trials and toxicology studies to find new markers of disease and adverse reactions. Scala media “It would be a great advantage (cochlear duct) to be able to perform proteomics analysis on samples where the outcomes of the patients are already known,” says David Speicher of the Wistar Institute. But formalin cross-links proteins to nucleic acids and other proteins, Spiral turning them into globs, and parligament µm affin imprisons proteins. “Since pathologists aren’t going to change their standard practice to meet the A photomicrograph of a paraffin-embedded cochlea goals of molecular biology, we have sample. to change molecular biology techniques to [accommodate] pathologists,” for extracting peptides from FFPE tissues ( J. Proteome Res. 2005, doi 10.1021/ says David Krizman of Expression pr050208p). After the paraffin is rePathology. moved with heptane, the tissue is susReasoning that mass spectrometrists pended in a mixture of SDS, ammonium typically disassemble proteins before anbicarbonate, and dithiothreitol (DTT). alyzing them anyway, several groups After a brief sonication, the mixture is have recently devised methods for reincubated at 70 °C for 1 h. The next leasing peptides from FFPE tissues, cirstep is to modify cysteines by adding cumventing the problem of extracting iodoacetamide, incubating the mixture in intact proteins. “It’s easier to extract peptides than intact proteins from FFPE the dark for 1 h, and adding DTT. The proteins in the tissue are then digested tissue because, even if one or a few sites with trypsin and analyzed by MS. “Ulon a given protein remain cross-linked trasonic disruption of the tissue is an imto other macromolecules or have been portant step because it breaks down the chemically modified, trypsin may be able to release multiple unmodified pep- bulk tissue, allowing trypsin to permeate more readily,” Palmer-Toy explains. tides from the remaining regions of the Palmer-Toy applied this method to protein,” Speicher says. soft tissue from a stenotic region of the In general, the researchers dissolve ear canal, fixing and embedding half the the paraffin with an organic solvent, resample and freezing the rest. In all, he move non-proteinaceous biological maidentified 573 different peptides from terials, and use a protease to cleave the 155 proteins. Whereas 20% of the proproteins into peptides. After identifying teins were unique to the frozen half, the peptides with MS, they use databas40% were unique to the FFPE half. es to identify the proteins from which “Our conjecture is that the frozen tissue unique peptides were released.
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searchers also compared mouse liver lobes that had been frozen with those that had been treated with formalin and encased in paraffin. The fresh-frozen tissue yielded 2001 unique peptides from 776 identifiable proteins, whereas the FFPE tissue yielded 1710 peptides from 684 proteins. “So we really think we have reached the benchmark set by frozen tissue,” says SAIC scientist Tom Conrads, who adds that the group has now efficiently extracted proteins from FFPE clinical samples that have been stored at room temperature for up to 15 yr. Speicher says he is impressed by this work because the researchers have reported the largest number of proteins seen from FFPE tissue samples and because the parallel comparisons of FFPE and frozen tissue showed surprisingly similar numbers of protein identifications with similar extents of protein coverage. “Furthermore, in their analysis of prostate cancer specimens and surrounding normal tissues,” he says, “the distribution of some relatively low abundance tumor-associated proteins, such as prostate-specific antigen, were detected in the expected regions of the specimens.” Karl-Friedrich Becker at the Technical University of Munich says that, in contrast to the other groups, he has developed a method for extracting fulllength proteins, rather than peptides, from FFPE tissue, but he’s unwilling to provide any details. “We are able to extract intact proteins that are compatible with a variety of assay formats—not only MS, but also western blotting and protein arrays,” he says. Applying this method to FFPE colon tissue, he identified four proteins at their expected molecular weights. Speicher says that it’s too early to assess the usefulness of the new extraction protocols because the reports are so preliminary. But a useful method, he says, “would allow you to dig deeply into the proteome and make at least semiquantitative comparisons.” a —Linda Sage
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sample of the same cells. Peptides from 263 proteins were common to both, but many proteins identified in the fixed sample were not seen in the fresh sample. “We believe that this is a result of formalin inactivation of endogenous proteases in the fixed sample,” Elenitoba-Johnson explains. The next set of studies, he says, will target clinical samples. The first step in Expression Pathology’s protocol is to cut standard 5–7-µmthick FFPE tissue sections onto glass slides and remove the paraffin with an organic solvent followed by a series of graded alcohols and rehydration in water. Next, the cells of interest are microdissected or needle-dissected and placed in a proprietary extraction buffer called Liquid Tissue MS buffer, which has been on the market since November 2004. The cells are then incubated at 95 °C for 90 min, and their proteins are digested overnight with trypsin. Disulfide bonds are broken down when the sample is placed in DTT and heated at 95 °C for 5 min. Researchers analyze the samples by MS. Timothy Veenstra and colleagues at Science Applications International Corp. (SAIC)-Frederick, who are working at the National Cancer Institute, collaborated with Expression Pathology to generate the protocol. Testing it on cells microdissected from FFPE prostate tissue (Mol. Cell. Proteomics 2005, doi 10.1074/mcp.M500102-MCP200), Veenstra’s group identified 1300 unique peptides representing 702 proteins from benign prostatic hyperplastic tissue and 2200 unique peptides representing 1156 proteins from prostate cancer tissue. Importantly, one of the latter was prostate-specific antigen. “The fact that we could see so many proteins in these samples even though we started with only tens of thousands of cells tells us that the extraction method is quite efficient and very robust for proteomics analysis by MS,” Veenstra says. To investigate the effect of fixation and embedding on protein yield, the re-
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