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Thermo Fisher Team Presents Mass Spec Assays for Cancer Signaling at AACR


NEW YORK (GenomeWeb) – Thermo Fisher Scientific researchers presented a study this week at the American Association for Cancer Research meeting in New Orleans detailing the company's mass spec-based workflows for measuring cancer cell signaling.

The work demonstrated the ability of multiplexed protein immunoenrichment coupled to targeted mass spec quantitation to measure panels of proteins and phosphoproteins key to cancer signaling.

Additionally, the research indicates that parallel-reaction monitoring mass spec can now approach the sensitivity of conventional selected-reaction monitoring assays while retaining advantages in terms of specificity and ease of assay development, said Bhavinkumar Patel, a senior research scientist in mass spec reagents and protein biology at Thermo Fisher Scientific and first author on a poster presented at AACR.

In the study, Patel and his colleagues used immunoenrichment followed by PRM mass spec on a Q Exactive HF and SRM mass spec on a TSQ Vantage instrument to measure levels of 12 proteins and 11 phosphoproteins in the IGF1R/AKT/mTOR signaling pathway, which is a key pathway in cancer and the development of drug resistance. They tested their assays in three cell lines, measuring expression of their protein targets before and after stimulation with IGF and comparing those measurements to ones taken using ELISAs, Western blotting, and Luminex immunoassays.

The study stemmed from a previous effort in which company scientists optimized immunoenrichment workflows for coupling to mass spec, identifying the ideal sets of materials and chemistries for use in IP-MS assays.

"The first question for us was whether we could optimize wash buffers and elution buffers in a way that they are mass spec compatible. If you can achieve this, after immunoprecipitation [of your protein targets] you don't have to run a gel and you can perform in-solution digestion before taking it straight into [the mass spec]," Patel said.

He and his colleagues presented such a workflow, which leverages upfront single and multiplex immunoenrichment to improve the performance of targeted mass spec analysis, at last year's AACR meeting and the workflow has since been commercialized through a set of kits with reagents and protocols for IP-MS experiments.

"In this study we choose a biologically important cancer pathway, and we utilize these kits to develop assays to look at the protein abundance of these pathway targets," Patel said.

They also wanted to benchmark their mass spec assays against conventional immunoassays, given that such assays are still more commonly used by cancer researchers — and life science researchers generally — than is mass spec.

In particular, Patel said, Western blots remain highly popular, despite their limitations.

"When we go to AACR especially, everyone is running Western blots," he said. "It takes a lot of time to do them, and we all know they are very semi-quantitative, they don't give you a good idea about absolute quantitation of your target proteins. So we wanted to see how [performance] differs between existing immunoassay techniques and mass spectrometry analysis."

Looking at two representative targets, total AKT and phosphor IGF1R, the researchers found good correlation between the Luminex, ELISA, and mass spec approaches, but poor correlation between those approaches and Western blotting, Patel said.

The researchers also looked at the performance of the mass spec assays separately, reporting that their method allowed for simultaneous quantitation of 12 total protein and 11 phosphoprotein targets in the low to sub-femtomolar range.

Particularly for the phosphoproteins, immunoenrichment was necessary, Patel said, noting that many of these are low-abundance targets that were not detected by mass spec assays without immunoenrichment.

The researchers used both PRM and SRM mass spec in the work, finding, Patel noted, that the two methods provided essentially the same level of sensitivity.

"In terms of sensitivity and the lower limit of quantitation, the values of SRM and PRM are very similar," he said. "A lot of those things have to do with what kind of peptides you are selecting, but in our hands both techniques give similar dynamic ranges and similar lower limits of quantitation."

This is noteworthy given that, while PRM has certain advantages compared to SRM, SRM has traditionally had the edge in terms of sensitivity.

PRM is essentially a variety of data-independent mass spec in which the mass spectrometer, rather than analyzing the full range of a sample, is trained on a more targeted mass and time window. The technique arose several years ago when researchers including Bruno Domon, head of the Luxembourg Clinical Proteomics Center, and Josh Coon of the University of Wisconsin, Madison, began exploring it on high-resolution instruments like Thermo Fisher Scientific's Q Exactive as an alternative to traditional triple quadrupole-based targeted protein quantitation.

The approach has several potential advantages. For instance, the larger number of product ions monitored via PRM should improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. The instrument's high resolution can also reduce the effects of co-isolating background peptides. And, because researchers are able to monitor a large number of ions instead of having to select the most appropriate ones for monitoring beforehand as in SRM, assay development can be much simpler.

Use of the technique has grown in recent years, but many researchers assumed that for maximum sensitivity, conventional SRM assays would remain the method of choice. For instance, in a 2012 interview with GenomeWeb, Christoph Borchers, director of the University of Victoria-Genome British Columbia Proteomics Centre and founder and chief scientific officer of targeted proteomics firm MRM Proteomics, called PRM "an interesting technology" but added that he had "sincere doubts that it can reach the sensitivity of a high-end triple quadrupole."

Early PRM development work was largely done on the first-generation Q Exactive instrument, however. And, Patel said, upon moving to the more powerful Q Exactive HF instrument, he and his colleagues have found PRM's sensitivity to be a match for SRM.

"There is a huge improvement with the Q Exactive HF," he said. "We validated all these assays on [that instrument] and we observed that we were achieving sensitivity similar to what people are getting with SRM."