SP – The Coon lab seems to prefer OMSSA. Why? Is it solely related to ETD performance, or does it extend to other fragmentation mechanisms as well?
Josh – OMSSA was the first algorithm to be compatible with ETD, which is what we initially started out doing almost exclusively. We also liked that it was free and we could put it on multiple servers without charge. As we began incorporating other fragmentation methods into our workflows, we tested the performance of OMSSA relative to other algorithms and found it to be comparable, if not superior. So since we were already invested in the pipeline, we decided why pay for something if it’s working just fine?
SP – Classical shotgun proteomics using a DDA mode may be reaching a plateau in terms of proteins identified. It takes a lot of effort, and especially MS acquisition time, to get deeper into the proteome. Could DIA modes such as SWATH represent a serious alternative?
Josh – I think the key thing to allow people to get deeper into the proteome is better separations. I don’t really think that SWATH or any type of mass spec-only technique is going to allow one to circumvent the separations to get the maximum gain. While the faster scanning afforded by techniques like SWATH can be helpful, at the end of the day, you’re only able to detect the ions that are presented to the mass spectrometer. If you don’t separate the peptides well, issues such as ion suppression preclude the detection of low-abundance peptides. So it seems to me that after separations the mass spectrometer can play a role but it’s not going to offer complete proteomic coverage in a matter of hours because there’s always that front stuff that’s still required.
SP – What could be the next big innovation in mass spectrometry? Could the idea of ion mobility be combined for further separation of co-eluting ions?
Josh – Again I think that separations are pretty important. Coming up with creative ways of how to control and use instrument bandwidth is important, improving ion capacity of mass analyzers, and well as increasing ability to isolate narrowly. I think improvements in all those areas could add a lot. Ion mobility is an interesting concept and certainly has its applications. But due to things like ion suppression and sensitivity issues I still don’t think it will be the magic bullet. Pre-ionization separations are key.
SP – Where do you envision quantitative proteomics being in 5–10 years? What methodologies do you think will be routinely used?
Josh – I think that we are increasingly able, over the last several years, to compare multiple things using primarily isobaric tagging. I think that there’s recent work and ideas that would indicate that those methods can be further “plexed” and expanded to making dozens or many dozens of simultaneous comparisons quite feasible. This type of throughput could allow for hundreds or even thousands of samples to be compared and I think that will help contribute to discoveries that are translational and clinically relevant. I think that’s where the field goes.
SP – What does the Biemann Medal mean to you?
Josh – Well the Biemann Medal is quite an incredible surprise and honor. It really means a lot to me. It’s great to see that the field recognizes our contributions to the development of ETD. It’s very rewarding to see that people have found the method useful. It’s really a great honor and I think that while the medal was awarded to a single person there are really a lot of people out there that contributed to make ETD what it is today and they should all be quite proud.
SP – What do you see as the future for ETD? How does ETD fit into the future of proteomics?
Josh – For a while the major thrust of proteomics method development was to increase detection. But since we are getting pretty good at detecting proteins I think we will see a shift from simple detection to deep characterization of proteins. And I think in this pursuit ETD really has a lot to offer. Some of the initial appeal of ETD was diminished by the advent of HCD, which was both compatible with isobaric tags and did a decent job of fragmenting higher charged and phosphorylated peptides. But I think that there are still certain PTMs-glycosylation, O-GlcNac, sulfonation, a handful of others-that are really only tractable with a method like ETD or ECD. Those methods haven’t really gotten the same amount of attention because they’re hard to enrich for. But Al Burlingame, for example, has recently demonstrated the detection of 6,000 O-GlcNac sites with ETD, and it looks like this modification is pretty widespread and very prevalent and one might expect that that field might become significant. If that’s true, ETD will do very well in it. Large peptides and intact proteins are also a growing field of interest and an area where ETD provides some definite advantages. Basically, I think ETD opens a lot of doors that were previously closed to scientists. For example, the reason that trypsin is still one of the better enzymes for proteomics is because it’s robust, it’s easy to get your hands on, it’s cheap, it’s very efficient, and peptides that are tryptic size tend to chromarograph very well, so all the separations that people have worked on the for the past 20 years are very effective for peptides that size. I think those are things that have limited the complete conversion of people doing collision activation to ETD. But with ETD we can explore other enzymes and begin to consider modifications that aren’t amenable to collisional activation. This work is already underway as exemplified by Al Burlingame’s O-GlcNac analyses and Albert Heck’s work with Lys-N. I just think it will take a little time to develop the new enrichment, chromatography, and digestion techniques that make full use of ETD’s capabilities.
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Joshua J. Coon
Department of Chemistry
University of Wisconsin – Madison
After receiving a PhD from the University of Florida Josh undertook a post doc with Don Hunt where he and John Syka invented electron transfer dissociation (ETD). As a professor at the University of Wisconsin he has continued his work on mass spec instrumentation by adding ETD capabilities to Orbitrap mass spectrometers, coupling a gas chromatogaph to an Orbitrap, and fixing a laser to a dual cell ion trap. He has applied these techniques to study human embryonic stem cells, post transcriptional regulation of protein levels in yeast, and plant phosphoproteomics. He was the recepient of the 2012 ASMS Biemann Medal which recognizes “significant achievement in basic or applied mass spectrometry made by an individual early in his or her career”
Phanstiel DH, Brumbaugh J, Wenger CD, Tian S, Probasco MD, Bailey DJ, Swaney DL, Tervo MA, Bolin JM, Ruotti V, Stewart R, Thomson JA, and Coon JJ Proteomic and phosphoproteomic comparison of human ES and iPS cells Nature Methods. 2011
Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS, and Coon JJ Gas-phase purifcation enables accurate, multiplexed proteome quantifcation with isobaric tagging Nature Methods. 2011
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