NEW YORK (GenomeWeb News) – Researchers at Moffitt Cancer Center are using multiple-reaction monitoring mass spec to build panels of protein biomarker assays for use in clinical cancer research.
Currently, the center is employing a panel composed of MRM assays to more than 80 proteins to track patient response to BRAF and HSP90 inhibitors in melanoma. And while technical challenges have to date limited the application of MRM mass spec to actual patient tumor samples, John Koomen, scientific director of proteomics at Moffitt, told ProteoMonitor this week that he believes the approach is "becoming more and more feasible."
Koomen and his colleagues presented the MRM panel they are using for their melanoma work in a paper published this week in Molecular & Cellular Proteomics. In the study – which the authors described as "proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic" – the researchers used the panel to investigate protein signaling changes in melanoma in response to treatment with inhibitors of HSP90 and MEK.
Among their findings was that HSP90 inhibition in melanoma cells resistant to the BRAF inhibitor vemurafenib (marketed by Genentech as Zelboraf) involves inhibition of signaling through the PI3K/AKT/mTOR pathway, with use of a combined PI3K/mTOR inhibitor partially reversing acquired vemurafenib resistance. They also identified the receptor tyrosine kinase PDGFR β as a potential mediator of BRAF inhibitor resistance and linked increased β-catenin expression following MEK inhibition to NRAS mutant melanoma cell survival.
Additionally, the researchers demonstrated that they could apply their panel to samples as small as 50μg of total protein, including fine needle aspirates from xenografts and clinical tumor specimens. Achieving sufficient sensitivity using the small sample sizes typically available from patient tumor specimens has been a key issue in the drive to use MRM mass spec for this sort of work.
Discussing the Moffitt team's efforts to overcome this challenge, Koomen noted that while advances in triple quadrupole instrumentation had helped, careful assay development was just as important. Indeed, in the MCP paper, he and his colleagues used relatively old triple quad models – Thermo Fisher Scientific's TSQ Quantum Ultra or Vantage.
"We've really taken to the LC-MRM approach as kind of a cottage industry where we are making assays one at a time" to cancer proteins of interest, Koomen said.
To build their assays, the Moffitt researchers isolate their proteins of interest in cell lines they expect to express them, then screen for proteotypic peptides they believe would work as good surrogates of their target, taking into account factors like isoforms, mutations, and post-translational modifications.
"Once we have that peptide we can synthesize [it] and characterize it to be sure that it matches what we saw in the original endogenous sample, and we can build [the assay] from there," Koomen said.
In past work, Koomen and his team have developed groups of MRM assays for investigation of pathways including β-catenin in colon cancer and NFКβ in Fanconi anemia, but the MCP paper represents their first effort to combine assays to several pathways in one experiment, he said.
Koomen compared their assay building effort to work with reverse phase protein arrays, in which researchers have built out panels of reagents for examining key cancer signaling pathways.
"We're taking a similar approach, except using a [mass spec] assay instead of an immunoassay," he said.
RPPAs have been used by a number of researchers for work similar to the Moffitt's MCP study looking at protein signaling and drug response in cancer samples. In perhaps the highest profile such study to date, researchers affiliated with breast cancer charity the Side-Out Foundation presented last June findings on the use of various omics data, including phosphoproteomic profiles developed via RPPA, in personalizing breast cancer treatment.
RPPAs share the potential drawbacks of all immunoassays – issues of antibody availability and specificity, for instance – but they hold significant advantages over mass spec in terms of sensitivity, a feature that can be particularly important when looking at dozens of proteins in small samples like clinical tumor specimens. For instance, while Koomen and his group used 50μg of total protein, RPPA can work with sample sizes in the range of the picograms to nanograms of protein lysate.
Given this, Koomen said that it's likely that RPPA will continue to outperform mass spec for some analyses, particularly, he noted, for clinical phosphoproteomic work where target peptides are often low abundance and fly relatively poorly in mass spectrometers. However, he noted, improvements in these workflows are under development.
In the meantime, MRM is "very good at measuring protein expression," he said, adding that he doesn't see "any barrier to using LC-MRM for protein biomarkers."
He noted, though, that the 80-plus protein panel presented in the MCP paper is more a research tool than a clinical assay. Ultimately, he said, the goal would be to use the panel to identify smaller sets of proteins relevant to guiding therapy in specific patients, which could then be turned into a clinical assay.
"It becomes very difficult both for clinical chemists and for physicians that are using these types of protein biomarker platforms to have [as many analytes as in the MCP panel]," Koomen said. "So it's really going to be critical to define the key points that you want to evaluate. It's a challenge and its something that is going to be constantly evaluated because what you have to really deal with in cancer is that the disease is very complex and very individual, and so you are going to have to have a personalized strategy."
In the Moffitt's melanoma trial, Koomen and his colleagues are using the panel to look at patients both before and after combination treatment with a BRAF and HSP90 inhibitor and, down the road, at patients post-relapse, as well.
In addition to developing standard triple quad-based MRM assays, Koomen said he is also looking into targeted quantitation on high-resolution machines – a practice that has grown in popularity in recent years.
"We recently got a [Thermo Fisher] Q Exactive, and I'm very interested in the parallel reaction monitoring [high-res quantitation] type of strategy," he said. "I think if you go to that sort of approach you can deal with your background interferences more readily, so you might be able to use shorter gradients and have faster run times."
In the MCP paper, the researchers used LC gradients in the 35 to 40 minute range.
Koomen noted, though, that in terms of taking assays into the clinic, triple quads had an advantage due to greater familiarity with these machines among clinicians and regulators.
"The triple quad has such a long history [in the clinic] that it seems straightforward to use it for protein biomarkers," he said. "But if you want to use some of these other techniques, maybe you're a little further away."