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MEETING NEWS Katie Cottingham reports from the 52nd American Nashville, Tenn.
ECD-like spectra without a magnet
Society for Mass Spectrometry Conference—
acquired by CAD, all you can really conclude is that it is a phosphopeptide,” says Melanie Schroeder, a graduate student in Hunt’s group. “You don’t know what Large, highly charged, and posttranslathe amino acid sequence is.” This probtionally modified peptides are difficult lem is not limited to de novo sequencto fragment by conventional methods, ing, says Schroeder. Random backbone such as collisionally activated dissociafragmentation is also necessary for search tion (CAD). Although electron-capture algorithms to successfully identify known dissociation (ECD) can handle these peptides. With ETD, fragmentation octypes of peptides, it has only been poscurs along the backbone, sible in very expensive which yields a clear amino FTICR MS instruments (a) 3+ acid sequence. Schroeder until recently (see story 50 (M+3H–H PO ) 3 4 CAD adds that because the phoson p 258 A). To over40 phate group is still bound, come these limitations, 30 one can pinpoint the exact Donald Hunt and col20 3+ residues that are phosphorleagues at the University (M+3H–2H3PO4) ylated. The Hunt group of Virginia and Thermo 10 also has preliminary eviElectron Corp. have de0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 dence that ETD works in veloped another fragmenm/z the same way on glycosyltation technique called ated peptides. electron-transfer dissocia(b) +++ ++• Hunt believes ETD will tion (ETD), which makes 50 (M+3H) (M+3H) ETD (precursor) change the way scientists ECD-like data accessible 40 conduct proteomics experion less-expensive ion trap +•• 30 (M+3H) ments. For example, trypsin instruments. c4 z8 c c1 20 8 cleaves proteins into many Hunt and other proc c c2 z6 z4 3 5 z1 y6 z7 c 10 small fragments that are teomics researchers comy z z y1 ++ a8 6c 5 7 2 z3 z7 6 about 10–15 amino acids monly study proteins on ion 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 long. Using trypsin to ditraps, but directly adapting m/z gest a complex protein mixECD for conventional 3-D ion traps would have been Spectra obtained for the same multiphosphorylated peptide with many basic ture, therefore, can result in thousands of peptides, quite a challenge, according amino acids using (a) CAD and (b) ETD. which can be a challenge to John Syka, a graduate student in Hunt’s lab and an employee of believe that the negative reports in the lit- to analyze. “Now, with this technology, erature were the final word. “But I had to you’re no longer limited to tryptic pepThermo Electron Corp. Therefore, Syka tides,” says Hunt. Additionally, Coon has convince John and Josh to spend a whole and Josh Coon, a postdoctoral fellow in year building an instrument to test it out,” performed initial experiments showing the the lab, adapted a radial ejection linear feasibility of using ETD for top-down prosays Hunt. “It’s to their credit that they ion trap for ETD. In ECD, thermal electrons react with hung in there, knowing full well that other teomics studies, in which a whole protein is fragmented within the mass spectromepeople had said it wouldn’t work.” peptides or proteins to cleave the bonds ter without prior enzymatic digestion. One important application of ETD is along their backbones, yielding c- and ETD opens up ECD-quality data the analysis of phosphorylated peptides in z-type fragment ions. Thermal electrons to scientists who do not have FTICR LC/MS/MS experiments. When phoswould not last long in the radio frequenMS instruments, which can cost up to cy field of an ion trap, says Syka, so in the phopeptides are subjected to CAD, most $1 million. “[ETD] is a really powerful of the fragmentation events occur at the new ETD method, the team uses negatechnique that will become accessible to phosphate groups instead of along the tive ions to deliver electrons to multiply anybody who uses ion trap-type instruprotonated peptides or proteins. After the peptide backbone. “So if you try to sit down with a pencil and paper to manual- ments, and that’s most people doing biopositively charged peptides and the negalogical MS,” says Coon. ly sequence a phosphopeptide spectrum tively charged anions are introduced into 260 A
A N A LY T I C A L C H E M I S T R Y / A U G U S T 1 , 2 0 0 4
COURTESY OF DONALD HUNT AND MELANIE SCHROEDER, UNIVERSITY OF VIRGINIA
Percent relative abundance
the instrument, they are trapped and allowed to react. Electrons are transferred to the peptide ions; this transfer creates radical species that dissociate in the same way as in ECD. Cleavage occurs along the peptide backbone and is relatively insensitive to residue type or position. ETD had been tried before, says Hunt, but no one had been able to make it work. He was undaunted, however, and didn’t
