This story is part of an ongoing series on emerging proteomic technologies. See the bottom of the article for a complete list of stories in the series. List will be updated as stories are added.
NEW YORK (GenomeWeb) – What will proteomics look like five years from now?
In a broad sense, the field will likely be much the same — a mix of mass spec and immunoassays, discovery and targeted workflows slowly but steadily improving in terms of their speed, sensitivity, accuracy, and throughput.
Looking more closely, though, what emerging technologies are most likely over the next half decade or so to make their way into the field's mainstream? What techniques currently being worked out in just a handful of labs are likely to gain wide acceptance among researchers and industry in the near future?
Are there, for instance, any methods currently out there with status analogous to, say, Swath-style data independent analysis mass spec in 2010 or 2011?
Scripps Institute researcher John Yates III was the first to investigate Swath-style DIA, introducing a version of it performed on a ThermoElectron LTQ mass spec in a 2004 Nature Methods paper. But mass spec instrumentation and software was not yet at a place where widespread implementation of the technique was practical, and it was not until AB Sciex introduced Swath on its TripleTOF 5600+ mass spec in 2012 that the method began to see significant uptake by the broader proteomics community. (Waters released its MSE DIA technique in 2006, but this method is based on a different workflow than Swath-based approaches.)
The technique was, five years ago, still little used save for in laboratories like that of Ruedi Aebersold at the Swiss Federal Institute of Technology Zurich, where it was being developed. Yet, it was on the cusp of a major commercial release and, since then, Swath-style DIA methods have exploded in popularity, with labs across academia and industry taking up the technique and a number of mass spec vendors offering varieties of the method on their machines.
With Swath's example in mind, GenomeWeb has surveyed a number of leading proteomics researchers to learn what they think are the currently emerging technologies that could similarly see dramatic growth in popularity and usage in coming years.
As might be expected, opinions vary, with the scientists surveyed citing a wide variety of possibilities. There was some overlap, however, with a several technologies or approaches highlighted by a number of researchers.
For instance, several of those surveyed highlighted as particularly promising the potential of mass spectrometry combined with techniques like cross-linking and hydrogen-deuterium exchange to aid in structural analysis of proteins and protein complexes.
"In the next couple of years I see a great uptake by the community in the techniques for mass spectrometry-based structural proteomics," said University of Victoria researcher Christoph Borchers, noting that such techniques could provide "structural information about proteins and protein complexes which are very difficult to analyze using common structural biology approaches like X-ray crystallography and NMR."
"The development of structural proteomics techniques has made significant progress in the last years and [these techniques] are now ready for prime time, meaning that they are useful to answer biological questions and solve biomedical problems," he added.
Alexander Makarov, director of global research LSMS at Thermo Fisher Scientific and inventor of the Orbitrap analyzer, likewise highlighted the potential of mass spec-based structural analysis, noting specifically the use of cross-linking combined with MS3-level analysis.
Makarov also cited the potential of middle-down mass spec, especially enabled by ultraviolet dissociation. This was likewise selected by Utrecht University researcher Albert Heck (Makarov also holds an appointment at Utrecht.)
While trypsin digest-based bottom-up workflows have dominated proteomics since the field's inception and, more recently, top-down, intact protein approaches have made significant advances, middle-down holds out the possibility of combining the best of both worlds, using digestion to break proteins into sizes suitability for mass spec analysis, but creating longer peptides than is done via trypsin, which simplifies analysis and allows for more complete sequence coverage.
Heck said he believes that in the next five years attention would turn in a significant way toward middle-down approaches.
"In [middle-down] the aim is to generate long peptides that can be fully sequenced by MS/MS," he said. "In this way the full sequence of a protein of average size of 40 kDa can be mapped by four peptides, instead of 30 to 40 tryptic peptides."
"Another advantage is that we should be able then to map co-occurring protein post-translational modifications," he added, noting that middle-down "represents, in my view, the best way forward."
Additional technology development is still needed, however, Heck said. For instance, either enzymatic or chemical methods for generating peptides of the desired length are still needed, as are new separation methods and new fragmentation and bioinformatics tools.
Bruno Domon, head of the Luxembourg Clinical Proteomics Center, suggested that use of high-resolution instruments like QTOFs and Orbitraps for targeted quantitation, rather than the more traditional triple quadrupoles is an area ripe for increased adoption.
Such methods, which Domon has been at the forefront of developing, offer potential advantages over conventional triple quad-based MRM assays in that they are able to target a particular precursor ion and then monitor not just a few, but all of the resulting product ions.
This means, among other things, that researchers don't have to determine upfront what the best transitions to monitor will be, significantly reducing assay development time. Additionally, the larger number of product ions monitored via high-resolution methods should improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. This might also reduce the effects of co-isolating background peptides.
"High-resolution quantitative work has certainly emerged in the last two years or so and has gained quite a bit of traction," Domon said.
Relatedly, SISCAPA Assay Technologies CEO Leigh Anderson said that he expected TOF-based quantitation would emerge as a key technology in the near future, particularly for clinical applications.
"The analytical world currently assumes that triple quads offer the best quantitation," Anderson said. "However TOF analyzers probably allow better precision in heavy:light ratio measurements — for example, peptide quant with a labeled internal standard — because they measure both ions in the same spectrum."
"MRM assays, for all their power, are always measuring the heavy and light peptides at different times, so any variation in the electrospray source will cause noise in the ratio," he said, noting that this was particularly important in the case of clinical measurements.
University of Washington researcher Michael MacCoss, meanwhile, suggested that the most analogous technology to Swath five years ago is, in fact, an improved version of Swath, along with other similar DIA methods.
"I personally believe that the future is in technologies that improve the selectivity of DIA without losing sensitivity," he told GenomeWeb. "We need the selectivity of 4 m/z [or smaller] windows across the entire mass range, and improvements in mass spectrometer speed won't get us there alone without sacrificing sensitivity."
"We have made large improvements in this area," MacCoss noted. "However, I don't think the entire set of tools to make use of these data will be available for wide adoption for another four to five years."
The above technologies represent just a sampling of the notions put forth by the surveyed researchers. Over the course of this year, GenomeWeb plans to explore the ideas and approaches offered in response to our original question in a series of stories taking a closer look at each of them individually.
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