细胞培养中氨基酸的稳定同位素标记
西妥昔单抗
下调和上调
表皮生长因子受体
信号转导
ERBB3型
MAPK/ERK通路
癌症研究
化学
生物
计算生物学
定量蛋白质组学
结直肠癌
癌症
蛋白质组学
细胞生物学
受体
生物化学
遗传学
基因
作者
Markus Stepath,Birgit Zülch,Abdelouahid Maghnouj,Karin Schork,Michael Turewicz,Martin Eisenacher,Stephan A. Hahn,Barbara Sitek,Thilo Bracht
标识
DOI:10.1021/acs.jproteome.9b00701
摘要
We evaluated the quantification strategies label-free (LF), stable isotope labeling by amino acids in cell culture (SILAC), and tandem mass tags (TMT) and their performance in quantification of proteins and phosphosites (p-sites) to identify the most powerful approach for monitoring cellular signaling. We analyzed the epidermal growth factor receptor (EGFR) signaling network, which plays an essential role in colorectal cancer, and studied its dynamics within 24 h upon treatment with the EGFR-blocking antibody cetuximab, representing the first cellular adaption toward therapy. LF achieved superior coverage but was outperformed by label-based approaches regarding technical variability, especially for quantification of p-sites. TMT showed the lowest coverage and most missing values. We found that its performance considerably decreases when experimental replicates are distributed over several TMT plexes. SILAC showed the highest precision and outstanding performance for quantification of p-sites, rendering it the method of choice for analyzing cellular signaling in cell culture models. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated. These dynamics represented an initial downregulation of the MAPK pathway, which was partially rescued as early as 24 h after treatment. We identified upregulation and signaling via ERBB3 as well as calcium and cAMP signaling as possible mechanisms bypassing the blockage of EGFR.
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