At next week’s Pittsburgh Conference in Chicago, Thermo Fisher Scientific will be highlighting one of its newest mass spectrometers equipped with electron transfer dissociation capability, a technique relatively new to proteomics research, but which proponents say carries the potential to significantly improve peptide data.
Launched in June at the American Society for Mass Spectrometry conference, the LTQ XL is an upgrade to the LTQ instrument and was the first linear ion trap mass spec on the market incorporating ETD technology. Other vendors, including Bruker BioSciences and Agilent Technologies, have launched 3D ion trap mass specs with ETD capability.
Since releasing the LTQ XL, Thermo has taken orders for more than 50 systems — which includes both upgrades to the LTQ mass spec and orders for the LTQ XL — and installed more than 20 units.
ETD was first devised in 2004 by Don Hunt’s laboratory at the University of Virginia. The inventor of the technology, John Syka, is a scientist with Thermo who took time out from his duties at the company to finish his PhD at the university.
The technique is just beginning to be used in the proteomics community where peptide fragmentation has been traditionally done with collision-induced dissociation. Supporters of the method say that it has a number of advantages over CID, including providing a more reliable and surer way of capturing phosphorylation sites. In large peptides, in particular, ETD provides better sequence coverage, proponents say.
In a recent study published in The Proceedings of the National Academy of Sciences, Johns Hopkins researchers compared ETD with CID and found that ETD identified 60 percent more phosphopeptides than CID, and 40 percent more fragment ions that facilitated localization of phosphorylation sites [See PM 02/08/07].
ETD combined with CID methods can provide researchers a greater depth of information about peptides than they would get otherwise, said Ian Jardine, vice president of global research and development for Thermo.
“The power of the ETD technique is that it provides totally complementary fragmentation information to the CID technique. So if you have a peptide, the CID technique tends to provide y- and b- ions, and the ETD technique, on the other hand, provides z- and c- ions,” he said.
“The most powerful experiment is to run both and then look at both sets of data … and that way you get a huge increase in the information content, and the accuracy, and certainty of identification of peptides,” Jardine said.
While ETD capability is available on 3D ion trap mass specs, Thermo says that the larger ion storage capacity and faster cycle times of a linear ion trap instrument allows researchers to alternate between ETD and CID on sample runs. Switching between the two modes is possible on a 3D ion trap mass spec with ETD capability, according to Jardine, but the slower speed of the 3D ion trap makes such switching difficult to manage.
“As the chromatogram with all of the peptides is eluting, you’re liable to miss a lot of peptides. Because of the speed of the LTQ, its actually recommended implementation is to switch rapidly back and forth between CID and ETD,” he said.
The platform can also use multiple other reagent ions, “in particular ions which induce proton transfer reactions, mainly proton abstraction, which are extremely powerful in reducing the complex charge state of very large peptides and small proteins,” Jardine said.
In the fall, Thermo released an updated version of its BioWorks software that streamlines the processing of ETD data. CID and ETD spectra are searched independently for results with or without phosphorylation and other posttranslational modifications for easy comparison and sorting, the company said.
“The most powerful experiment is to run both and then look at both sets of data … and that way you get a huge increase in the information content, and the accuracy, and certainty of identification of peptides.”
At least one researcher who is currently working on further developing the ETD technique, however, said the verdict is still out on whether a linear ion trap ETD system is a significant improvement over other ETD-enabled mass specs.
Akhilesh Pandey, the senior author on the PNAS article and a user of the Agilent 6340 3D ion trap mass spec with ETD capability, said that while a linear ion trap mass spec has greater ion trap capacity and faster cycle times, he and other researchers will want more data generated by the research community verifying Thermo’s claims.
“Many people in this business wait for about one year before there is accumulated data and wisdom from the user side about what they are finding,” he said.
Meanwhile, Thermo is working on developing other types of mass specs with higher resolution incorporating ETD, but said that such technology is a long way off.
“Technically, it’s quite difficult, but eventually we will be able to do that, I’m sure.” Jardine said. “Because of the success of the LTQ FT [Fourier transform MS] and the LTQ Orbitrap [hybrid MS], people clearly find the additional capability very powerful, and so that’s a kind of obvious thing for us to do. I just caution the time frame is not determined because technically, it’s not trivial,” he said.
A method similar to ETD, electron capture dissociation, is already being used on Fourier transform mass specs, Jardine added.
It is unclear where the rest of the industry stands on developing linear ion trap instruments with ETD capability. Waters does not have a linear ion trap mass spec and does not have plans to introduce one, said a company spokesman. Agilent Technologies did not respond to a request for comment. Bruker BioSciences and Applied Biosciences declined to comment.
An LTQ XL with ETD costs about $400,000.The older LTQ platform can be upgraded for ETD capability for about $100,000.