NEW YORK(GenomeWeb) – The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has contracted researchers to develop targeted mass spec assays for proteins in the RAS signaling pathway as part of an initiative launched by the National Cancer Institute.
Announced last month, the project aims to develop multiple-reaction monitoring mass spec and immuno-MRM mass spec assays for roughly 100 proteins involved in RAS signaling, John Koomen, scientific director of proteomics at Moffitt Cancer Center and one of the participating researchers, told GenomeWeb.
Despite the importance of RAS signaling, coverage of the pathway by targeted proteomic assays is fairly spotty, Koomen said.
"There's not a lot available out in the literature," he said. "My lab has published a few things looking at protein expression in multiple components of these pathways, and there are a few other publications out there that focus on individual proteins and their post-translational modifications. But to try to develop as comprehensive a platform as this that is biologically driven hasn't really been done."
Other researchers being funded through CPTAC for the RAS work include Steven Carr of the Broad Institute and Amanda Paulovich of the Fred Hutchinson Cancer Research Center, both CPTAC participants. Gordon Whiteley at the NCI's Antibody Characterization Program is leading development of antibodies for the immuno-MRM assays.
The group "has a fairly broad spectrum of targets," Koomen said. "Some of them are in that central canonical MAP kinase signaling pathway, and others are a little bit more disparate." The NCI has listed the project's initial 100 targets here.
The goal is to develop both MRM and immuno-MRM assays to the targets, he said, noting that while a number of the proteins will likely be detectable using conventional LC-MRM, lower-abundance molecules will likely require immuno-MRM.
Beyond that, Koomen added, immuno-MRM will likely be required to reach the project's goal of multiplexing the full complement of assays.
"We're at the point of being able to multiplex the assays together for what we hope in the end will be a single mass spectrometry analysis of all the targets," he said, noting that antibody enrichment of target peptides prior to MRM analysis will help account for different protein abundance levels while also providing cleaner samples with less interferences.
Peptide-level immuno-MRM – or SISCAPA (for Stable Isotope Standards and Capture by Anti-Peptide Antibodies) was pioneered in large part by proteomics researcher Leigh Anderson, who founded the firm SISCAPA Assay Technologies to commercialize the method. The technique has advantages over conventional MRM in that the immunoenrichment step improves the sensitivity of the mass spec instrumentation while also cleaning up the sample so as to allow for shorter LC run times and, therefore, higher throughput – a key consideration in clinical research.
Another potential advantage, Koomen noted, is in transferring assays across different sample types. Different samples can present different interfering molecules, and so researchers need to check for interferences when using an assay developed in one sample type in a new type.
"That is one of the other reasons immuno-MRM is so attractive," Koomen said. "When you digest to the peptide level, the amount of non-specific binding you get is significantly lower than if you were trying to enrich these proteins with antibodies at the protein level. And I think they will ultimately be very transferable from one sample type to another."
One potential limiting factor, however, is that the technique requires a quality antibody to each target peptide, making method development considerably more labor and time intensive than conventional MRM.
Koomen suggested that the RAS assay project was in large part "a test case for whether the NIH and NCI want to develop a large number of reagents for cancer-relevant proteins for immuno-MRM."
The NCI has in recent years funded efforts focused on developing anti-peptide antibodies for immuno-MRM. For instance, in 2012, the agency announced it would be awarding four $200,000 Phase I and four $1 million Phase II SBIR grants for development of such antibodies. Awardees were to develop at least 10 anti-peptide antibodies in Phase I and at least 100 anti-peptide antibodies in Phase II.
In June 2013, the agency released the first set of antibodies developed by its Antibody Characterization Program specifically for immuno-MRM, a set of 15 rabbit monoclonals.
Last February, OriGene Technologies said it received a contract from the NCI to develop antibodies for use in SISCAPA-style assays. AvantGen and Precision Antibody were also awarded SBIR grants through NCI for generation of anti-peptide antibodies in 2014.
CPTAC has made considerably more progress in development of standard MRM assays, launching in August 2014 its MRM assay portal, which contains MRM mass spec assays for 456 different peptides and 282 proteins.
While the RAS work is largely focused on development of triple quad-based MRM assays, Koomen said his team would be looking into using high-resolution instruments – Thermo Fisher Scientific's Q Exactive, specifically – to develop some assays as part of the project.
For example, "we have a few assays, for very long peptides that aren't going to be effectively monitored [using standard MRM] because it would be a challenge to predict or select fragment ions that would give you good sensitivity," he said. Using a high-resolution instrument would allow the researchers to monitor many fragment ions at once, allowing for easier assay development in cases where appropriate ions are difficult to predict.
"So we do have some pilot projects for Q Exactive instruments we'll attempt as part of the project," Koomen said.
He estimated the effort to build the roughly 100 standard and immuno-MRM assays would take around two years.