DISTILLATION AND
EVAPORATION OF HEAT SENSITIVE MATERIALS • MOLECULAR DISTILLATION A CONCENTRATION
DISSECTING HISTONES FROM THE TOP DOWN Method combines mass spectrometry and database searching to characterize histones
A FRACTIONATION • EVAPORATION
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For the analysis, Kelleher pulled out the EIL L. KELLEHER WANTS TO DO big gun of mass spectrometry: quadrupole proteomics in a "fundamentalFourier transform mass spectrometry ly different way" Rather than (quadrupole FTMS). FTMS uses a big sudigest proteins with enzymes perconducting magnet, making it "the before starting an analysis, Porsche of mass spectrometers," Kelleher Kelleher, an assistant professor of chemremarks. 'As if that's not enough, we stick istry at the University of Illinois, Urbanathis quadrupole on the front, and it makes Champaign, takes the intact protein and the system even better. It enables us to enlets a mass spectrometer do the work. He hance low-abundance signals." FTMS and calls his approach, which he helped pioquadrupole MS rely on different types of neer with Fred McLafferty his graduate mass analyzers. adviser at Cornell University, top-down mass spectrometry Once the protein is in the mass spectrometer, Kelleher and his students break "You take the intact molecule, as prethe protein into smaller pieces using a sented by the biological source, and you method called electron capture dissociadon't degrade it with enzymes," Kelleher tion. A lot of fragment ions are produced, says. That allows you to study "all these complex molecular forms of the proteins that are expressed." Histones are a prime example of proteins whose modifications affect their function. D N A wraps around histones, which serve as part of the packaging for the chromosomes in the cell nucleus. "There are multiple forms of this protein, and it's really important in D N A binding and genome packaging and gene transcription," Kelleher says. The posttranslational modifi- H I S T O N E H U N T E R S Kelleher (second from left) cations on the exposed "tails" and his students (from left) Taylor, Pesavento, and form part ofwhat some people Kim use top-down mass spectrometry and shotgun call the "histone code," which annotation to identify modifications in histones. may be involved in deteiminKelleher says. "We take this horrendousing which genes are being transcribed. ly complicated data, and we use it to query Kelleher and graduate students James a horrendously complicated database. J. Pesavento, %ng-Bin Kim, and GregoThe key is high mass accuracy for the fragry K. Taylor combine a mass spectrometment ions." ric approach with "shotgun annotation" That database is filled with predictions to identify the modifications on human histone H 4 {J.Am. Chem. Soc, 126, 3386 of all the possible combinations of modifications. Limiting the predicted fragments (2004)]. Shotgun annotation involves cre"by reasonable biochemistry" resulted in a ating a database of various protein forms database of about 50,000 possibilities. that could be generated from combinato"We combinatorially consider all the rial consideration of different protein possibilities," Kelleher says. "Now we modifications. have a technology—shotgun annotation "We started with the simplest histone," and all this fancy mass spec—to take this Kelleher says. There arefivemain histones in humans. H 4 has seven well-known mod- very complex data and query this complex database. Out pops the right anification sites, which follow set rules for swer— automatically." their modifications.
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For H4, the method generated 91 fragment ions, 78 of which matched a particular prediction in the database. Kelleher believes that the other fragments didn't match because they were not within the tight mass accuracy of the database search. Kelleher relies on the quality of the data to make sure the search algorithm correctly determines which modifications are present on a particular histone. "There's no magic wand here," he says. "Ifyou have good data, you can have an automated characterization, but you're always limited by your data." Good data can differentiate the subtle gap between the right answer and the next-best hit. Kelleher believes that top-down mass spectrometry makes the process of histone characterization "cleaner" than it is with other approaches. "In bottom-up methods, the peptides that are created are from a mixture of different protein forms. You garble all the different forms together in this digest and lose the connectivity of which peptides came from which protein form," he says. "The beauty of top-down MS is that you first measure these different forms and their ratios. Ifou get a picture of the histone code at the intact level." IN THE FUTURE, Kelleher plans to use top-down MS and shotgun annotation to track how the histone modifications vary during the cell cycle. He also hopes to pinpoint where in the genome particular histone forms are located. "You want to know if a gene is actively being transcribed in vivo, what are the histone modifications at that site in the genome? What's the code? That's where we're headed." Alma L. Burlingame, the director of the mass spectrometry facility at the University of California, San Francisco, says, "Kelleher's method will facilitate global regio assignment of multiple modifications to individual protein species." He also believes, though, that more detailed sequencing done by another fragmentation method known as collision-induced dissociation will pinpoint modification sites in instances where electron capture does not cleave the protein in enough places. Nevertheless, Kelleher is evangelistic about proteomics using top-down MS. He has created a website for top-down proteomics with a protein database that emphasizes posttranslational modifications using a program called ProSight PTM. His website currently has about 100 users. "Top down is going to have a role to play in proteomics, and that role is going to do nothing but grow," he says.—CELIA HENRY HTTP://WWW.CEN-ONLINE.ORG
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