Targeted mass spectrometry-based assays enable multiplex quantification of receptor tyrosine kinase, MAP Kinase, and AKT signaling.

Abstract

SUMMARY:

A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX-4720. These assays replace over 60 Western blots with quantitative mass spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism of action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov.

MOTIVATION:

A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 Western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor.

• Authors: Jeffrey R Whiteaker, Kanika Sharma, Melissa A Hoffman, Eric Kuhn, Lei Zhao, Alexandra R Cocco, Regine M Schoenherr, Jacob J Kennedy, Ulianna Voytovich, Chenwei Lin, Bin Fang, Kiah Bowers, Gordon Whiteley, Simona Colantonio, William Bocik, Rhonda Roberts, Tara Hiltke, Emily Boja, Henry Rodriguez, Frank McCormick, Matthew Holderfield, Steven A Carr, John M Koomen, Amanda G Paulovich
• Journal: Cell reports methods (Cell Rep Methods) Vol. 1 Issue 3 (07 26 2021) ISSN: 2667-2375 [Electronic] United States
• PMID: 34671754 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural)

Chemical References

• Proto-Oncogene Proteins c-akt
• Receptor Protein-Tyrosine Kinases
• Mitogen-Activated Protein Kinase Kinases
• Tyrosine

Topics

• Humans
• Proto-Oncogene Proteins c-akt (metabolism)
• Mass Spectrometry (methods)
• Receptor Protein-Tyrosine Kinases
• Melanoma
• Mitogen-Activated Protein Kinase Kinases
• Tyrosine