Meeting News: Looking at large peptides ... - ACS Publications

Meeting News: Looking at large peptides. | Proteomics study of signaling networks. | Criteria for Publication of Proteomics Data. Universal proteomics...
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M E E T I N G Katie Cottingham reports from the American Society for Mass Spectrometry 53rd Conference on Mass Spectrometry—San Antonio, Texas. Looking at large peptides Many proteomics researchers study either intact proteins with a top-down MS approach or small tryptic peptides with a bottom-up MS strategy. But Barry Karger and colleagues at Northeastern University have developed a new method, called extended range proteomic analysis (ERPA), in which large peptides are analyzed by ion trap/Fourier transform (FT) MS ( J. Proteome Res. 2005, 4, 1155–1170. The researchers obtain increased sequence coverage in addition to more data about posttranslational modifications (PTMs) with ERPA than with traditional methods. In the ERPA approach, proteins are digested with Lys-C, although other en-

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zymes can be used. “Because you’re only cutting at one site with Lys-C, most of the time you’ll find at least one extra basic amino acid in the fragment than you would for trypsin, which leads to a 10-fold or higher increase in sensitivity,” says Karger. He explains that the additional basic amino acid results in better ionization of the peptide, which is particularly important when the peptide carries a negatively charged PTM, such as a phosphate group. After digestion, the peptides are separated by liquid chromatography and analyzed by MS. According to Karger, his group’s new ion trap/FTMS instrument gave them a unique opportunity to study large peptide fragments. He says that the high mass resolution capability of the FT mass spectrometer allows one to determine the charge states of large peptides; this is difficult to do with other MS instruments. At the same time that the FT part determines

the charge states, the ion trap part of the hybrid instrument automatically performs MS2 and MS3 sequentially on the highest intensity ions. The researchers have demonstrated the ERPA method with two proteins, casein and epidermal growth factor receptor (EGFR), which is a cancer biomarker. All known glycosylation and phosphorylation sites were identified. Karger and colleagues obtained 97% sequence coverage of -casein and 95% sequence coverage of EGFR. Karger says that the group has recently increased the sensitivity of the ERPA method for comprehensive characterization of transmembrane glycoproteins, such as EGFR, down to the low-femtomole level. Instead of using a wider-packed conventional capillary, Karger incorporated a 20-µm-i.d. monolithic column, which he and his colleagues developed previously (Anal. Chem. 2003, 75, 5306–5316).

Proteomics study of signaling networks Blagoy Blagoev, Matthias Mann, and coworkers at the University of Southern Denmark and Odense University Hospital (Denmark) have used a quantitative proteomics method called stable isotope labeling by amino acids in cell culture (SILAC) to dissect signaling pathways involved in human mesenchymal stem cell (hMSC) differentiation (Science 2005, 308, 1472–1477). The researchers identified both overlapping and distinct members of two receptor tyrosine kinase pathways that trigger osteoblast formation. “When we started working with MSCs, we were surprised that very little was known about the regulatory effects of growth factors on these cells,” says Blagoev. So the researchers added epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor, and fibroblast growth factor to hMSCs. These growth factors bind to cellular receptors that phosphorylate themselves and other proteins. The researchers immunoprecipitated proteins from the cells with antibodies to phosphotyrosine and found that EGF and PDGF triggered the strongest phosphorylation responses. When EGF and PDGF were added to hMSCs that were induced to differentiate, however, only EGF enhanced os-

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No bones about it. EGF enhances osteoblast formation in hMSCs that have been induced to differentiate. Cells were stained with Alizarin Red S dye to visualize mature osteoblast mineralization. (Adapted with permission. Copyright 2005 American Association for the Advancement of Science.)

teoblast formation. “We wanted to compare the global signaling networks . . . to see if we could find out what could trigger these differential effects,” says Blagoev. The researchers used the SILAC proteomics method to study the pathways. Control cells were given normal arginine, but those treated with EGF and PDGF were given different isotopic variants of arginine. The cellular lysates were mixed, immunoprecipitated with phosphotyrosine antibodies, and analyzed by liquid chromatography/MS. EGF and PDGF stimulated the phosphorylation of many of the same substrates, but EGF uniquely activated eight proteins and PDGF uniquely activated nine proteins. For example, all of the regulatory and catalytic subunits of phosphoinositide 3-kinase (PI3K) were

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present in high amounts only in the tyrosine-phosphorylated complexes of PDGF-treated cells. When the researchers blocked PI3K by adding Wortmannin to differentiating hMSCs that were treated with PDGF, bone formation was greatly enhanced. The cells resembled those treated with EGF. Therefore, Blagoev says that PI3K is a control point that determines the differential effects of EGF and PDGF on osteoblast formation. According to Blagoev, the researchers plan to continue work on the EGF and PDGF pathways and to apply the method to additional signaling networks to determine other control points. “We hope that in this way, you could direct more precisely [how cells differentiate] for optimal use in stem cell research,” he says.

Katie Cottingham reports from the American Society for Biochemistry and Molecular Biology (ASBMB) Workshop: Criteria for Publication of Proteomics Data —Paris, France. Universal proteomics guidelines debated What do you get when you put 30 bioinformaticians, instrument vendors, journal editors, and proteomics scientists in a room for 2 days? Organizers of the ASBMB workshop on criteria for publishing proteomics papers hoped to create a set of guidelines that would be universally accepted by the scientific community. A draft of the document was hammered out at the workshop and was made available on July 14 for public comment. The group used a set of criteria, often referred to as the MCP guidelines (Mol. Cell. Proteomics 2004, 3, 531–533), as a framework on which to build the universal guidelines. Steve Carr, who is at the Broad Institute and is associate editor of MCP, says that the journal’s editors created the MCP guidelines because erroneous identifications were seeping into the literature. “The understanding of how [proteomics] software works or what the criteria are for acceptable performance of the instrument are not widely understood,” he says. “People from the biological sciences are coming in and using these things—and rightly so—but lack in many cases a firm grip on what’s acceptable and what’s not.” The Paris workshop was a way to include additional stakeholders and produce a document meant for the entire proteomics community, says Carr. On the first day, attendees presented talks

on diverse topics, including the HUPO Proteomics Standards Initiative, current quantitative proteomics methods, and a summary of a previous National Institutes of Health proteomics standards meeting. Attendees got down to the nitty-gritty on the second day by huddling together in small subgroups to focus on different aspects of the guidelines. After much discussion and a welldeserved lunch break, the chairs of the subgroups presented new, revised criteria for their assigned topics. A few differences emerged between the MCP-published guidelines and the new universal draft. For example, the draft includes a section on quantitative proteomics, which was missing from the other document. The section on posttranslational modifications was also expanded. Ron Beavis, who is with Beavis Informatics and is an editorial board member of JPR, adds, “The other kind of overall difference is that . . . this new document gives authors a much clearer view of what it is that they are expected to have in a manuscript.” Participating editors from Proteomics, MCP, JPR, and the Nature journals stated that they would consult with their staffs and editorial boards before signing on to the draft. Editors expressed concern about the final section of the MCP guidelines, which encouraged researchers to deposit data into a repository. They said that such a guideline should only be enforced once a community-accepted standard is established. As a beta test for a repository of MCP data, Karl Clauser of the Broad Institute volunteered disk space at his institution. Since the meeting ended, participants have been emailing each other to discuss refinements. Once the final ver-

G O V E R N M E N T Protein Structure Initiative The Protein Structure Initiative (PSI), which is funded by the National Institute of General Medical Sciences and the National Center for Research Resources, has entered its second phase. With the announcement of 10 new PSI research centers, the emphasis now shifts from methods development to the application of those methods to determine thousands of protein structures from

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sions were assembled into a single draft document, Ralph Bradshaw, who is at the University of California, Irvine, and is editor of MCP, says that he emailed it to researchers who are on various proteomics mailing lists and on journal editorial boards. Anyone who is interested but did not receive a copy by email can also review the draft on the MCP and Proteomics websites or contact Bradshaw at [email protected]. Comments are due to Bradshaw by October 15. He will then turn comments over to the subgroup chairs for consideration. He hopes that the final set of universal guidelines will be adopted by major proteomics journals by early 2006. William Hancock, who is at Northeastern University and is editor-in-chief of JPR, says that a subcommittee of JPR editorial advisory board members is working on a similar set of guidelines, which will be posted on the journal’s website in the near future. Will proteomics researchers and editors really accept a universal set of guidelines? The document “only has value if people are willing to adopt it,” says Bradshaw. Every journal is different, and editors are free to mold the guidelines to fit their needs, he says. Researchers who don’t adhere to the universal criteria may face difficulties publishing their work if the guidelines are adopted by most journals. Because the proteomics field is constantly evolving, Bradshaw says he will assemble a committee, initially composed of the workshop’s subgroup chairs, to keep an eye on new issues that will needtobeincludedin the future. As Beavis says, “I think this shouldn’t be looked at as the last word—rather, as the start of the process.”

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several organisms, including humans. The new production phase includes two types of centers. Four large-scale research centers will use high-throughput methods and instruments developed during the initial pilot phase to produce up to 4000 protein structures over 5 years. Six specialized centers will focus on developing methods to crack the structures of a few hard-to-study proteins, such as those on cell membranes and those in

complexes with other molecules. All of the information produced by the PSI will be deposited in the Protein Data Bank (PDB) (www.rcsb.org/pdb), a public repository of 3-D protein and nucleic acid structures funded by the National Institutes of Health and the National Science Foundation. Researchers can access structures and other relevant data at the PDB to help them understand protein functions and shapes.

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