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. 2021 Jul 26;1(3):100015.
doi: 10.1016/j.crmeth.2021.100015. Epub 2021 Jun 14.

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

Jeffrey R Whiteaker  1 , Kanika Sharma  2 , Melissa A Hoffman  3 , Eric Kuhn  4 , Lei Zhao  1 , Alexandra R Cocco  5 , Regine M Schoenherr  1 , Jacob J Kennedy  1 , Ulianna Voytovich  1 , Chenwei Lin  1 , Bin Fang  3 , Kiah Bowers  3 , Gordon Whiteley  6 , Simona Colantonio  6 , William Bocik  6 , Rhonda Roberts  6 , Tara Hiltke  7 , Emily Boja  7 , Henry Rodriguez  7 , Frank McCormick  2   8 , Matthew Holderfield  2   9 , Steven A Carr  4 , John M Koomen  3 , Amanda G Paulovich  1
Affiliations

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

Jeffrey R Whiteaker et al. Cell Rep Methods. .

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.

Keywords: AKT; Cancer signaling; MAP kinase; RAS; RTK; assay resource; immuno-MRM; pharmacodynamics; quantitative proteomics; targeted therapy.

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Conflict of interest statement

DECLARATION OF INTERESTS The authors declare no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
Development of quantitative assay panels targeting cancer signaling to promote cellular growth and proliferation (A) RTK, MAPK, and AKT signaling networks were targeted for MS-based assay development to quantify expression and phosphorylation of proteins that drive cellular growth and proliferation in cancer. Proteins targeted by the MRM assay panels are colored blue; additional signaling nodes not included in the assay panel are shown in gray; the BRAF inhibitor, PLX4720, is shown in red. (B) The different sample processing workflows culminate in LC-MRM of tryptic peptides using a spiked-in stable isotope-labeled standard (SIS) for each analyte. Direct-MRM targets higher-abundance proteins, IMAC-MRM targets phosphopeptides (i.e., pSTY) for enrichment prior to MRM, and immuno-MRM uses custom monoclonal antibodies for peptide immunoaffinity enrichment of selected unmodified and phosphorylated peptides. Peptides measured in common between methods are shown in the Venn diagrams. The protocols, reagents, and assay characterization data, as well as demonstration of utility of the methods for pharmacodynamic and proof-of-mechanism studies, are presented in this article.
Figure 2
Figure 2
Unsupervised clustering of quantitative MRM data groups primarily by biological differences in cell types (A–B) Unsupervised clustering of protein expression measured by direct-MRM (A), phosphorylation measured by IMAC-MRM (B), and protein expression and phosphorylation measured by immuno-MRM (C) show that protein expression and phosphorylation predominantly cluster by cell line with secondary clusters grouped by response to PLX treatment. Row heatmap values are Z score of the median response for each peptide analyte using the log2 transformed peak area ratio (light/heavy) values from the MRM data (n = 2 biological replicates); missing values were imputed with LLOQ values.
Figure 3
Figure 3
MRM assays show quantitative changes in signaling in melanoma and colorectal cancer cell lines after PLX4720 treatment Heatmaps of selected quantitative MRM measurements of proteins and phosphosites to examine central nodes of MAPK (RAS-RAF-MEK-ERK) signaling and demonstrate previously published mechanisms of BRAFi resistance in four cancer cell lines. MRM results shown are from immuno-MRM assays with the exceptions of p-EGFRY1092 and p-CTNB1S552 (which are IMAC-MRM results). For MRM assays, p-AKT1S473 and AKT2 peptide LLPP were chosen for correlation with pan AKT1/2/3 western blots (WB). Each cell in the heatmap is colored according to normalized values for individual analytes across all samples. The quantitative values were correlated with analysis by WB (STAR Methods). Analyte nomenclature was based on the sequence of the peptide analyzed by MRM (left side, “MRM assay” label) and the reported WB antibody specificity (right side, “Western blot” label). Vinculin was used as a loading control for WB. Bar plots are the mean of duplicate biological replicates. Error bars show the range of duplicate biological replicates.
Figure 4
Figure 4
Quantitation of paradoxical ERK1/2 activation after PLX4720 treatment in colorectal cancer and melanoma cell lines illustrates the molecular detail accessible through immuno-MRM measurements (A) Relative quantitation of ERK1/2 peptides are plotted as mean peak area ratio (PAR) comparing the light endogenous with the heavy internal standard in cell lines ± PLX4720. Error bars show the range of duplicate biological replicates. Dotted line shows the approximate lower limit of quantification (LLOQ). (B) Densitometry values from western blots (WB) of ERK1/2 expression and phosphorylation in the same cell lysates were used for correlation with immuno-MRM analysis. (C) The relative levels of total protein expression, plotted as the PAR for the peptide ALDLLDR, are related to levels of the phosphorylated p-ERK1Tyr204. The amino acid residue numbers of the peptides within the protein are shown in brackets. Samples are color-coded according to cell line and ±PLX4720 in the key at the top of the panel; data were labeled with time point.
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