NEW YORK (GenomeWeb) – Using C$1.2 million ($1.1 million) in funding from Genome BC and Genome Canada's Genomic Applications Partnership Program, proteomics firm MRM Proteomics (MRMP) and the University of Victoria-Genome BC Proteomics Centre are developing panels of multiple-reaction monitoring mass spec assays for quantifying mouse proteins.
According to Christoph Borchers, director of the Proteomics Centre and chief scientific officer at MRM Proteomics, the assays will focus on areas including cardiovascular disease and cancer and will be released as a product by MRM around the end of the year.
While MRM mass spec assays are widely used in proteomics research and are perhaps the primary tool for mass spec-based clinical proteomics, few such assays for mouse proteins are commercially available, Borchers said.
Indeed, he told ProteoMonitor that he was not aware of any commercial MRM assay panels for measuring proteins in mouse plasma, a situation that he noted was rather surprising given the popularity of mice as a model organism for biological research.
As with protein analysis more generally, antibody-based methods are commonly used to measure mouse plasma proteins, Borchers said. However, he noted that the relatively small volume of blood in a typical mouse limits the number of proteins that can be measured without killing the animal.
"A mouse has only around 1.4 ml of blood, so how many ELISAs can you do?" he said. "In most cases, you have to kill the mouse to get [all the blood] out."
This, in turn, makes longitudinal monitoring of mouse proteins impossible, something that Borchers said MRM panels could enable.
Using mass spec "we can detect hundreds of proteins out of 5 microliters," he said. "We can just take a bit out of the tail [periodically], and that way you can study the mouse over a long period of time."
Borchers had no explanation for why, given their potential utility as research tools, mouse plasma MRM panels had yet to make it to market, but, he said that he had received inquiries from potential customers even before news of the project was officially released this week.
"We've already gotten interest from pharma companies," he said. "They immediately called us up and said, 'As soon as the assay is ready, please let us know. We would like to have it.'"
Developing the mouse panels has not proven entirely straightforward, though, Borchers noted, citing the example of the mouse CVD panel he and his colleagues are working on.
In building the panel, the researchers are using strategies such as taking human CVD markers and looking for related proteins in mice, he said. However, he noted, this has proven a relatively difficult process.
"We have problems with detecting [the mouse proteins]," he said, adding that this could be due to a number of issues – for instance, that the mouse and human proteins are not related as they assumed, or that their detection is complicated by differences in mouse and human plasma.
"There are some technical difficulties we have with mouse plasma compared with human plasma," he said. "We probably need to optimize our digestion procedure, our cleanup procedure. We don't know [what the problem is] right now, but we can't detect [in mouse] many proteins that are detectable in human plasma. We don't know if that is intrinsic – that's what [the protein expression] is – or if these are technical problems."
Nonetheless, Borchers said, MRM expects to release the first set of assays around the end of the year. In addition to the CVD panel, the company is working on cancer marker panels, as well as assays for measuring proteins in specific tissues beyond plasma.
In human plasma, MRM is able to put together assays using one-dimension chromatography that can detect around 190 proteins in a single experiment and assays using two-dimensional chromatography that can detect around 300 proteins in an experiment, he said.
MRM is building the assays on Agilent's 6490 triple quadrupole instrument. The company is also expecting within the next several weeks delivery of Agilent's new 6495 triple quad, which, according to Agilent, offers around a five-fold increase in sensitivity compared to the 6490.
Beyond the assays themselves, MRM also offers detailed SOPs and high levels of technical support for customers, Borchers said, noting that a study he and his colleagues recently did as part of the Human Proteome Project demonstrated how important this sort of support can be.
In the study, they sent MRM assay kits to 16 labs around the world and found that while the kits performed well regardless of the mass spec platform used, results varied significantly depending on the experience of the technicians performing the assays.
Even in his own lab, Borchers said, results vary depending on the experience of the researcher running the assay.
"Here a technician did this for the very first time and he quantified around 20 proteins with a CV better than 20 percent, and then the second time it was up to 30 proteins," he said. "So you have to build up your skills."
To a large extent, he noted, this means basic manual steps like pipetting that ultimately will be eliminated by automation.
"That is the best way," he said. "Because these are not very fancy steps. In the future everything will be done with liquid handling systems. You will buy your LC-MS system along with a liquid handling system and it will come with a protocol and you'll upload it and then you'll run it and that will be it."