NEW YORK (GenomeWeb) – Researchers at Memorial Sloan Kettering Cancer Center have developed a mass spec workflow capable of measuring peptides in biological samples at zeptomolar concentrations.
According to Alex Kentsis, a pediatric oncologist at MSK and leader of the effort, the researchers are now implementing a CLIA version of the method that they plan to use in upcoming clinical trials to measure cancer signaling activity with the ultimate aim of guiding patient treatment.
Described in a study published last month in Molecular & Cellular Proteomics, the project began as an effort to determine just how sensitive a high-resolution, high mass accuracy trapping instrument like an Orbitrap could be compared to beam instruments like triple quadrupoles, which, Kentsis noted, are the traditional workhouses for quantitative mass spec.
The researchers investigated this question on Thermo Fisher Scientific's Orbitrap Fusion Hybrid instrument, which combines a quadrupole with an Orbitrap and linear ion trap.
"We had this new instrument, and, of course, with any new instrument you want to benchmark its performance," Kentsis said. "And we were surprised to find that, actually, the sensitivity that we could obtain exceeded all kinds of conventional measures."
Ultimately, he said, they were able to achieve "not quite single-molecule detection, but [detection] in the hundreds of molecules." Using a sample of synthetic peptides in solvent fed directly into the mass spectrometer, the researchers were able to achieve "yoctomolar sensitivity with more than seven orders of linear quantitative accuracy," they wrote, a level of detection uncommonly low enough that, Kentsis said, he had to look up the proper prefix to describe that concentration.
As would be expected, this level of detection didn't hold in actual biological samples, however, Kentsis said, he and his colleagues were still "pleasantly surprised" by the instrument's performance in complex samples like cell extracts where it was able to quantify molecules at the zeptomolar level.
"That is still far in excess of what we were expecting to see," he said, noting that typically researchers have managed zeptomolar detection in purified samples and attomolar detection in complex samples.
Using the workflow, the researchers were able to quantify the transcription factor MEF2C and assess its phosphorylation levels in 1 μg of extracts from 10,000 human cells at levels as low as 100 molecules per cell.
One key to achieving these levels of sensitivity was use of the instrument's ion trapping ability to accumulate target ions, increasing the total number of ions routed to the analyzer and, ultimately, the sensitivity of the analysis.
This "is a very classic, old idea," Kentsis said. "People have experimented with concentrating ions like this for a long, long time as part of designing ever more sensitive and accurate mass spectrometers."
The approach's effectiveness in the MCP study was due significantly to the high ion transfer efficiency of the instrument," he added. "It's just a very high-efficiency ion transfer device where a large fraction of the ions that are being delivered [into the mass spectrometer] actually end up being analyzed."
Also key is the purity of the ions being accumulated and delivered to the mass analyzer, Kentsis said, noting that without sufficient ion purity "contaminants would fill the mass analyzer and then dominate the detection and obscure the analytes of interest."
In fact, the authors wrote, "in consideration of the excellent transmission efficiency and detector sensitivity of current state-of-the-art mass spectrometers, co-accumulation of contaminants is thus the principal factor limiting sensitivity of targeted methods leveraging ion traps."
To improve ion purity, the researchers worked to optimize the upfront chromatography using a two-dimensional LC system that Kentsis said was essentially the same as other commonly used in proteomics work but automated to improve reproducibility and efficiency, thereby reducing coelution of contaminants and increasing the number of targeted measurements possible per sample.
"We're using established techniques, widely accessible, widely available techniques, but just done in a robust, reliable way, on a computer-controlled chromatograph," he said. "We've seen advances made in many fields through robust automation, and this is just an implementation of that concept for chromatography."
The decision to automate the chromatography also stemmed from the researchers' desire to use the workflow for clinical cancer work, Kentsis said. "We are working on clinical tests to measure signaling in cancer specimens as part of diagnostic clinical tests, and so that's why we decided to work on improving the sensitivity and quantitative accuracy [of the method] and automating it, because ultimately clinical tests need to be very robust and very reproducible."
He said the researchers plan to use the approach to quantify cancer signaling proteins and their activation levels to help improve diagnosis and guide patient therapy. In concept, this work is similar to antibody-based work by researchers like MD Anderson Cancer Center's Gordon Mills, who has used reverse-phase protein arrays to profile cancer signaling pathways in tens of thousands of patients.
Kentsis suggested, though, that mass spec might provide even better data. Historically, mass spec assays have not been sensitive enough to do the sort of profiling RPPA allowed for on small patient samples, but assays like those in the MCP paper offer the possibility of more quantitative measurements than antibody assays and the ability to profile pathways more broadly, covering molecules for which there are not good antibodies, Kentsis said.
"We can begin to look at specimens diagnostically with much higher precision," he said.
Kentsis said he and his colleagues are currently working to obtain New York State certification to use their assay for diagnostic purposes, after which they plan to begin using it in clinical trials, most likely starting in 2018. The project, he said, is funded in part by the National Cancer Institute's Cancer Moonshot initiative.