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Nonlinear's New Software Expands Protein Identification Beyond 2D Gels Into LC-MS

In a nod to the growing influence of liquid chromatography-based separations methods, Nonlinear Dynamics recently launched software that can enable researchers to use liquid chromatography/mass spectrometry analysis to identify and quantify proteins and peptides.
According to Nonlinear, the software, called Progenesis LC-MS, represents part of a new strategy for proteomics research at the company.
“Typically, people tend to use one [method] or the other,” Nonlinear CEO Will Dracup told ProteoMonitor recently. “What we’re trying to do is enable them to do both.”
Though Nonlinear has sold software for gel-based analyses for almost 20 years, Progenesis LC-MS is the first from the company designed specifically for liquid chromatography-based research.
The launch of the LC/MS software follows last year’s introduction of Progenesis SameSpots for 2D gel. An updated version of SameSpots was also introduced at the recent Association of Biomolecular Resource Facilities annual conference, along with the LC/MS software and Progenesis Stats, a multivariate statistical tool.
Together, the company said, the three products enable scientists to use both gels and liquid chromatography for protein identification research.
“From our point of view, this set of releases is just the first airing of a strategy that we’ve been working on for several years now, which is all about trying to [give] proteomics experiments the reliability that we think they’re capable of but which certainly haven’t been [achieved] very often,” Dracup said.
According to Nonlinear, the overlap in identified proteins between 2D gel and LC methods is as little as 10 percent. One study conducted by Helmut Meyer, director of the Medical Proteome Center in Ruhr-Universität Bochum, Germany, and others published in the January issue of the Journal of Proteome Research identified a 5-percent overlap between 2D PAGE and 3D LC-MS/MS.
Dracup said this suggests that both gel and LC methods should be used for protein identification.
Expanding LC Base
Founded in 1989, the company’s first commercial product was software for 2D and 1D gels for protein analysis. Since then, Nonlinear, based in Newcastle upon Tyne, UK, has continued developing software for gel-based methods.
However, in recent years, liquid chromatography methods have gained popularity as a separations method, particularly in the United States, though gel-based work is still the dominant method in Europe and Asia.
Nonlinear saw the shift as a market opportunity.
David Bramwell, technical director for Nonlinear, said that the company’s customer base is still mostly European and that its customer growth rate is larger in Europe than in the US. But, he said, “We’re expecting that to change with the LC product because that fits the demographics of the US. The US has gone very mass spec-based.”
There are many reasons for this, according to Aran Paulus, manager of R&D for the laboratory separations division of Bio-Rad Laboratories, which is doing strong 2D gel business in Europe and Asia. Among them is the notion that 2D gels are perceived as being very difficult, resulting in industry turning away from such methods because the chances for making mistakes run high due to the numerous manual steps involved, he said.
There is also a fascination with technology in the US, “and when you first hear of LC/LC-MS/MS, it sounds automatic, at least when it’s described in talks,” Paulus said.
In Europe and Asia, he added, researchers believe that separation on the protein level is more effective than separation on the peptide level, and “there is also a better level of understanding the technique.”
Dracup said, however, that Nonlinear did not launch Progenesis LC-MS specifically with the US market in mind.
“We released the [Progenesis LC-MS] because the analysis is very, very challenging,” he said. “When you talk to people, they’re using Mascot [from Matrix Science] to identify the peaks in the spectra, and then based on those identifications, matching up the different peaks and the different spectra, so they can then get an idea of what’s changed and what hasn’t changed.

“From our point of view, this set of releases is just the first airing of a strategy that we’ve been working on for several years now, which is all about trying to [give] proteomics experiments the reliability that we think they’re capable of but which certainly haven’t been [achieved] very often.”

“If you’re trying to use identification as a basis for deciding what to quantitate, then our premise on this front is what you need to be doing is actually work out which of the proteins or peptides that you need to identify and then really concentrating on them so you can take the time to get the identifications right,” Dracup said.
Building on SameSpots Technology
Progenesis LC-MS is based on the same image-analysis algorithm as its 2D software product. According to Nonlinear, the algorithm, called SameSpots, can extract more information from 2D gels — and now LC-MS — via improved image-alignment methods and background subtraction and normalization methods.
Pulling together the 2D gel and LC analysis is the Progenesis Stats software.
“Once we’ve done this initial stage of improving the quality of the data coming in, it opens the floodgates for using a lot more powerful statistical techniques and bringing things in like multivariate analysis,” Bramwell said.
The three software products used as a package facilitate quantitation as well as identification of proteins, Bramwell said.
Having the same outlines stabilizes low-level proteins “because with small expression proteins, adding a single pixel in or adding very few pixels can actually be a relatively high percentage of its volume, so you’ll find that the low expression spots were noisier when you used different outlines just by the fact that they had slightly different area inclusions,” Bramwell said.
“So if you use the same outline, you’re essentially unifying the sampling, and it drops the noise,” he said. Afterward, a researcher can also use other schemes such as variance stabilization and normalization to further drop the noise.
In addition to its UK headquarters, Nonlinear has an office in Durham, NC. In total, the company has about 30 full-time employees. Dracup declined to disclose any financial information about the privately held company.
He added that Nonlinear is in talks with other companies about possible collaborations, though he did not elaborate.
Since its formation 18 years ago, the company has weathered the ups and downs of the proteomics marketplace. While 2001 and 2003 were boom years, the period since has been more challenging, Dracup said. Other software competitors have ventured beyond their initial core business to try their hands at doing discovery work, but Dracup said Nonlinear’s business model is firmly embedded in the software domain.
“We think competing with our customers is wrong for us and is not where our skills lie, anyway,” he said.

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