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Proteomics gets out of a fix
SAUMIL MERCHANT, MEEI
about misidentifying peptides modified extracting peptides from FFPE tissues. during fixation, so we used very strinAfter the paraffin is removed with hepLike manuscripts in a vault, disease-regent identification criteria and estitane, the tissue is suspended in a mixlated proteins are locked away in the vast mated our misidentification frequency ture of SDS, ammonium bicarbonate, collection of formalin-fixed, paraffinby using a reversed protein database,” and dithiothreitol (DTT). After a brief embedded (FFPE) tissues stored by the he adds. sonication, the mixture is incubated at world’s pathologists. If only these proPalmer-Toy says his group’s proto70 °C for 1 h. The next step is to modify teins could be liberated, scientists could col is likely to work with a wide range of cysteines by adding iodoacetamide, indelve into the remains of clinical trials FFPE tissues. He explains, “We don’t see cubating the mixture in the dark for 1 h, and toxicology studies to find new markthat there is going to be any additional and adding DTT. The proteins in the ers of disease and adverse reactions. challenge posed by other tissue types, FFPE tissue are then digested into pep“It would be a great advantage to be considering that some of the materials tides with trypsin and analyzed by MS. able to perform proteomics analysis on we work with, including calcified samples where the outcomes of tissues, are generally the most difthe patients are already known,” ficult to work with.” According to says David Speicher of the WisPalmer-Toy, the group’s next chaltar Institute. lenge will be to extract proteins But formalin cross-links Scala media from celloidin-embedded tissues. proteins to nucleic acids and (cochlear duct) At the University of Utah, Kojo other proteins, turning them Elenitoba-Johnson removes the into globs, and paraffin imparaffin from diced paraffin prisons proteins. “Since patholblocks with xylene (Lab. Invest. ogists aren’t going to change 2005, 85, 1405–1415). After their standard practice to meet hydrating the sample in graded the goals of molecular biology, ethanols, he washes it in a mixwe have to change molecular Spiral ture of Tris HCl, Triton X-100, biology techniques to [accomligament µm sodium deoxycholate, sodium modate] pathologists,” says chloride, and ethylenediamineDavid Krizman of Expression Unlocked secrets. A photomicrograph of a paraffin-embedded tetraacetic acid. The proteins are Pathology. cochlea sample. then digested with trypsin or gluReasoning that mass tamic C endopeptidase and anaspectrometrists typically disaslyzed by MS. Testing this protocol on a “Ultrasonic disruption of the tissue is semble proteins before analyzing them 3-year-old FFPE cell block of a human an important step because it breaks anyway, several groups have recently lymphoma cell line and using the critedown the bulk tissue, allowing trypsin devised methods for releasing peptides rion of at least 2 unique peptides, he to permeate more readily,” Palmer-Toy from FFPE tissues, circumventing the identified 324 proteins, compared with explains. problem of extracting intact proteins. 514 in a frozen sample of the same Palmer-Toy applied this method to “It’s easier to extract peptides than cells. Peptides from 263 proteins were soft tissue from a stenotic region of the intact proteins from FFPE tissue becommon to both, but many proteins ear canal, fixing and embedding half the cause, even if one or a few sites on a identified in the fixed sample were not sample and freezing the rest. In all, he given protein remain cross-linked to seen in the fresh sample. “We believe identified 573 different peptides from other macromolecules or have been that this is a result of formalin inactiva155 proteins. Whereas 20% of the prochemically modified, trypsin may be tion of endogenous proteases in the teins were unique to the frozen half, able to release multiple unmodified fixed sample,” Elenitoba-Johnson 40% were unique to the FFPE half. “Our peptides from the remaining regions of explains. The next set of studies, he conjecture is that the frozen tissue lost the protein,” Speicher says. says, will target clinical samples. some proteins because endogenous In general, the researchers dissolve The first step in Expression Patholproteases may have been activated the paraffin with an organic solvent, ogy’s protocol is to cut standard 5–7when we processed it, just as they beremove non-proteinaceous biological µm-thick FFPE tissue sections onto come activated when you tear up letmaterials, and use a protease to cleave glass slides and remove the paraffin tuce and it becomes mushy,” Palmerthe proteins into peptides. After identiwith an organic solvent followed by a Toy says. He also analyzed a piece of fying the peptides with MS, they use series of graded alcohols and rehydraspiral ligament from a cochlea that had databases to identify the proteins from tion in water. Next, the cells of interest been fixed and embedded 4 years earwhich unique peptides were released. are microdissected or needle-dissected lier, identifying 125 proteins. “We were In this issue of JPR (pp 2404–2411), and placed in a proprietary extraction particularly pleased with the high seDarryl Erik Palmer-Toy and colleagues buffer called Liquid Tissue MS buffer, quence coverage that we observed for of the Harvard Partners Center for which has been on the market since proteins of interest,” he notes, cautionGenetics and Genomics, Massachusetts November 2004. The cells are then ining against putting too much stock in General Hospital, and the Massachucubated at 95 °C for 90 min, and their the total number of proteins “identified” setts Eye and Ear Infirmary (MEEI) proteins are digested overnight with in the various reports. “We were worried describe a nonproprietary method for
Journal of Proteome Research • Vol. 4, No. 6, 2005
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G O V E R N M E N T 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
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and embedding on protein yield, the researchers 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 ambient 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,
PSI creates MIAPE: Gel Electrophoresis
“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.” —Linda Sage
changes will be made to the text. The draft has just been reviewed internally by the PSI group and is now being tested On October 18, 2005, members of the HUPO Proteomics with real experimental data. Once this process is completed, Standards Initiative (PSI) posted a new minimum informathe draft will be sent to external expert reviewers and then pretion about a proteomics experiment (MIAPE) document, sented to the community as MIAPE: Gel Electrophoresis, vercalled MIAPE: Gel Electrophoresis, version 0.6, on the sion 1.0, at the Spring 2006 PSI meeting, which will be held PSI website (http://psidev.sourceforge.net). The document April 21–23 in San Francisco, Calif. is the latest in the family of MIAPE modules designed to MIAPE: Gel Electrophoresis only help researchers fully describe how includes information about an electrothey set up and performed proteophoresis experiment after the sample mics experiments. has been prepared and before image Frank Gibson at the University of analysis. Thus, the PSI members plan to Newcastle upon Tyne (U.K.) explains, produce additional modules that will “Protocols continue to increase in address these topics, called MIAPE: complexity as both methods and techProteomics Sample Processing and nologies evolve, so standard minimum MIAPE: Gel Informatics. According to reporting requirements for proteomics Gibson, the PSI group also intends to experiments . . . are required to facilidevelop data models like mzData, which tate the analysis, dissemination, and Context clues. The MIAPE: Gel Electrois used for MS data, for sample processexchange of data.” For example, he phoresis module will include details about ing, gel electrophoresis, and gel inforsays, the position of a spot on a 2DE how gel experiments were performed. matics. The gel electrophoresis model is gel could be an important piece of already under development. Gibson says that all of the data data, but it is meaningless unless one also knows informodels are created with the same functional genomics experimation about the composition of the gel and the running ment (FuGE) principles. FuGE “is a general structure for conconditions. Gibson adds that the document can provide cepts common to most functional genomics experiments; this guidance to journals about what types of information framework . . . will [allow] us to have interoperability across any should be included in a research paper. functional genomics experiment,” says Gibson. He adds that After Gibson and other PSI members wrote the systems biology approaches to data analysis will be easier to MIAPE: Gel Electrophoresis document, they posted it for implement because of this common structure. comment on the PSI website. Gibson says that the com—Katie Cottingham ments have been positive and that only a few minor
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