Many will remember 2003 as the year of biomarker hype and hope, but as a several researchers in the field told ProteoMonitor this month, biomarkers were not the only show in town — nor are they the only story to look for in 2004. Fourier-transform mass specs, an increased emphasis on quantitation and post-translational modifications, as well as the mass production of antibodies also featured prominently in 2003, and are slated to continue to play a central role in 2004.
FT-MS: here today, still here at ASMS?
FT-MS for the mass proteomics audience arrived last year in two forms: the LTQ-FT linear ion trap/Fourier-transform hybrid from Thermo Electron and the Q-q quadrupole Fourier-transform hybrid from Bruker BioSciences, both of which debuted at Pittcon. Nearly all the researchers interviewed for this article named FT-MS as one of the most interesting new toys to enter the mass spec market — yet most also agreed that the jury is still out on how FT-MS will fare in the long run.
Matthias Mann, professor of biochemistry and molecular biology at the University of Southern Denmark, called Thermo’s LTQ-FT a “potentially revolutionary development,” while Gary Siuzdak, director of the Center for Mass Spectrometry at Scripps Research Institute, took a more skeptical standpoint. “I think it will be useful, but it won’t be of critical importance — it won’t be this huge leap that we’re hoping for,” Siuzdak said. While FT-MS may be very sensitive, Siuzdak noted that its slow speed makes it impractical as an MS/MS workhorse. And even its advertised ultra-high resolution and mass accuracy has yet to be validated, Siuzdak said. “ASMS  will be a good indicator of what people are reporting on how these instruments are performing — how sensitive they actually are as opposed to the manufacturer specifications,” he said.
Assuming FT-MS turns out to be as robust as Mann hopes it will be, 2004 will the year for it to mature, he said. In 2004, FT-MS “has to be developed and put into protocols,” he said. As it seeps into more academic institutions, there should be plenty of opportunity for that to happen — and even more so as the price, still too high for most academics at $750,000 a pop for the cheapest version, continues to fall.
“It’s clear that FT-MS is going to be the way to go, and it seems like companies are now selling these machines much cheaper than even a year ago, so I suspect you’re going to see a spread of that technology,” said Michael Snyder, professor of molecular, cellular and developmental biology at Yale. Snyder said that while his lab did not have an FT-MS, Yale was likely to purchase one soon.
The linear ion trap, which also arrived last year in the form of Thermo’s LTQ stand-alone and Applied Biosystems’ 4000 Q-TRAP, was another development leaders in the field cited. It offers “the speed and dynamic range that you’re looking for,” Siuzdak said. He put a linear ion trap/TOF hybrid on his wish list for new tools in 2004.
Proteomics Bursts its Seams
In 2003, not only the instrumentation, but the core definition of proteomics changed in significant ways. “A few years back, proteomics was understood as running 2D gels and identifying the proteins separated by mass spec,” said Ruedi Aebersold, ICAT inventor and co-founder of the Institute for Systems Biology. “That has changed [in a way that] very much accelerated over the last year.” This change, Aebersold and others said, meant that last year ushered out once and for all the era of protein lists and began the era of quantitation, post-translational modifications, protein-protein interactions, and protein complexes. The work in these areas has only just begun.
“There’s been a lot of activity in the last couple of years [with post-translational modifications], but I don’t think there’s been much progress,” said Paul Tempst, director of the Protein Center at Memor-ial Sloan-Kettering Cancer Center. “You [only] look at the most abundant proteins, and you find 300 modified proteins out of the 5,000 — there’s got to be a better way.” Tempst also pointed out that most of the work so far concentrated only on phosphorylation, which is by no means the only modification — as Young-Ki Paik, secretary-general of HUPO and president of the Korean chapter of the organization, emphasized. “The phosphorylation problem seems to be holding some promise because some people have developed identification tools and things like that — but glycosylation is the biggest problem,” Paik said. To address this void, Paik said that 2004 would herald a Japanese launch of a glycoproteome project under the HUPO umbrella. “We are really hoping to have some kind of breakthrough in glycoproteomics separations or identifications,” he said.
Beyond this increasing focus on modifications and quantitation, the year just ended also widened the horizons for what proteomics can do. As an example of the ‘new’ proteomics, Snyder and Aebersold pointed to the work of Jonathan Weissman and Erin O’Shea at the Howard Hughes Medical Institute to create a TAP tag for and to localize each of the proteins in yeast (see PM 11-7-03). “These are particularly interesting studies because they show proteomics [to be] much larger than using a particular type of technique to do specific measurements on a lot of proteins,” Aebersold said. Pushing the envelope still further, Hochstrasser said to watch out in 2004 for the arrival from ABI of a kit for the molecular scanner he has been working on with the company for the past few years (see PM 4-15-02, 12-5-03). The scanner would produce an annotated, multidimensional image of a given gel. Hochstrasser indicated that the kit may also encompass direct tissue imaging, a project that ABI has been working on with Richard Caprioli at Vanderbilt University (see PM 7-1-02, 12-5-03). “Our brain is built to look at images,” Hochstrasser said. “Like the revolution of having a CT and MRI scan for medicine, obviously on a molecular side, if you can get a lot of images about a lot of the molecules in a sample as an image, then people will be fond of that.”
How Many Antibodies can you Make?
One of the most notable movements coming from HUPO in 2003, according to several of the researchers, was the planning of efforts to make antibodies to all proteins in the human body — a feat that, while difficult, could help push forward the development of protein arrays and the study of post-translational modifications, they said. “A world proteomics effort can only succeed if you have reproducible reagents, if you have consistency,” Tempst said. “One of the few things to have consistency is … monoclonal antibodies. That’s the only thing that can be propagated on the scale where you can distribute antibodies to everyone in the world who wants it.” Tempst said that in addition to antibodies against all the proteins encoded in the human genome, he would also like to see antibodies made against modifications.
Snyder also expressed interest in the antibody effort, which he predicted would “scale up” in 2004. His lab recently profiled antibody specificity and found that — as most antibody scientists would have predicted — monoclonal antibodies would be more suitable for the undertaking than polyclonal ones. But he added that high-throughput production of monoclonals may not be practical. “We may need aptamers as the way to go,” he said.
Hancock also pointed to specificity and the potential for high-throughput production as obstacles to such an antibody project, and then added the issue of cost. “Getting contract companies to generate antibodies is not cheap,” he warned.
Still, the potential pay-off in having a bank of antibodies available for use in developing a technology like the protein array — the progress of which is currently hampered by a lack of available antibody content — could be a valuable approach, according to Aebersold. How soon arrays become commonplace, he said, “depends on how quickly the antibodies can be generated and that, of course, takes time.”
So far there is no timetable for the completion of a total antibody bank, but “going forward, there is a definite trend towards expanding the repertoire of capture agents,” HUPO president Sam Hanash told ProteoMonitor in an e-mail.