Paulovich - Ras_assay_development

Targeted mass spectrometry-based assays for multiplex quantification of receptor tyrosine kinase, MAP Kinase, and AKT signaling
Data License: CC BY 4.0
  • Organism: Homo sapiens
  • Instrument: TSQ Quantiva,QTRAP 5500
  • SpikeIn: No
  • Keywords: Cancer signaling, immuno-MRM, assay resource, quantitative proteomics, pharmacodynamics, targeted therapy, RAS, RTK, AKT, MAP kinase
  • Submitter: Jeff Whiteaker
Abstract
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.
Experiment Description
Proteins and phosphorylation sites that sustain cell growth and proliferation in the RTK, MAPK, and AKT signaling networks were identified as targets for assay development by members of the US National Cancer Institute’s RAS Initiative (https://www.cancer.gov/research/key-initiatives/ras). Peptides amenable to mass spectrometry (MS) were identified for direct LC-MRM, IMAC-MRM, and immuno-MRM assays. Fit-for-purpose validation was performed to characterize each assay panel. We conducted proof-of-principle experiments to demonstrate application of the quantitative multiplexed assays in profiling changes in protein expression and phosphorylation in melanoma and colorectal cancer (CRC) cell lines harboring either BRAF or RAS mutations +/- PLX-4720 treatment.
Sample Description
We used four cell lines, including BRAF inhibitor-sensitive A375 (BRAFV600E) and -resistant SK-MEL-2 (NRASQ61R) melanoma cell lines, as well as resistant HT-29 (BRAFV600E) and HCT-116 (KRASG13D) colon cancer cell lines, as a test case in drug sensitivity and resistance based on their response to BRAF inhibition with 3 µM PLX-4720. Cell lysates (DMSO vehicle control or PLX-4720 treatment, harvested at 1.5, 24, and 48 h of drug exposure; two biological replicates) were proteolyzed with trypsin, and peptides were quantified directly by LC-MRM or used for enrichment prior to IMAC- or immuno-MRM analysis
Created on 10/12/20, 2:59 PM
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IMAC-MRM_Cell_Lysates_PLX4720_2020-10-12_13-59-14.sky.zip2020-10-12 14:55:4935491024421170
LC-MRM_Cell_Lysates_PLX4720_2020-10-12_14-03-00.sky.zip2020-10-12 14:55:4929120251989560
immuno-MRM_Cell_Lysates_PLX4720_Site2_Plex2_2020-10-12_14-10-36.sky.zip2020-10-12 14:55:491946925905446
immuno-MRM_Cell_Lysates_PLX4720_Site1_Plex1_2020-10-12_14-19-42.sky.zip2020-10-12 14:55:491461131499540