Dataset Information

Multilevel Omics-Readouts of Perturbation Studies in vitro are Determined by Memory Effects from Subculture

ABSTRACT: Mass spectrometry-based omics technologies are of increasing relevance for drug discovery. In order to map drug treatments to biological pathways for mode of action (MoA) deconvolution, they rely on the determination of multiple testing corrected significant molecular events. The main contributors to statistical significance are the effect size and the variation of a given molecule during the drug treatment. While the former is largely determined by the biological system and the impact of the drug, the latter reflects the robustness of the experimental workflow. The impact of sample preparation and instrumentation on variation are well characterized, but the impact of cell culture conditions on variation of omics readouts is less explored, specifically related to subculture conditions during cell expansion. Here, we provide evidence that memory effects from subculture directly influence the variation of multilevel omics readouts, including oxylipins and fatty acids, proteins and phosphorylated sites, and phenotypic parameters, e.g. cell motility. When comparing drug treatments after homogeneous and heterogeneous subculture, the coefficients of variation (CVs) of controls increased from 1% to 3% for oxylipins and fatty acids, 11% to 23% for proteomics, 24% to 53% for phosphoproteomics and 6% to 14% for cell motility, respectively. The number of identifications remained comparable and the fold-change distributions were also largely similar after both subculture conditions. Therefore, the decrease in CVs seems to be the main contributor to the increased number of significant molecular events in drug treatments after homogeneous subculture. This in turn increased information content in the obtained perturbation network, based on proteomics and phosphoproteomics data, from 58 to 321 causal conjectures. Perturbation networks after homogeneous and heterogeneous subculture allowed for delineating the established MoA of arsenic trioxide in detail, including enhanced kinase signaling and reduced MYC-related gene expression, induction of heat shock proteins and cell cycle disruption. However, only the perturbation network after homogeneous subculture enabled the discovery of unprecedented drug effects, including down-regulation and quadruple dephosphorylation of the transcription factor RB1, among others. In summary, memory effects from subculture have broad relevance for MoA testing in drug discovery since this impact clearly manifests in supernatants, whole cell lysates and phenotypic properties and its control enables a considerably enhanced MoA deconvolution, facilitating discovery efforts.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colonic Cancer Cell Line, Sw-480 Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Christopher Gerner

LAB HEAD: Christopher Gerner

PROVIDER: PXD039768 | Pride | 2025-04-22

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	22-Phos-0530_BC6_1_9139.d.rar	Other
	22-Phos-0531_BC7_1_9140.d.rar	Other
	22-Phos-0532_BC8_1_9141.d.rar	Other
	22-Phos-0533_BD1_1_9143.d.rar	Other
	22-Phos-0534_BD2_1_9144.d.rar	Other

Items per page:

1 - 5 of 50

Publications

Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

Bortel Patricia P Hagn Gerhard G Skos Lukas L Bileck Andrea A Paulitschke Verena V Paulitschke Philipp P Gleiter Lion L Mohr Thomas T Gerner Christopher C Meier-Menches Samuel M SM

Proceedings of the National Academy of Sciences of the United States of America 20240708 29

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence ...[more]

PMID: 38976734

Similar Datasets

Project description:Mass spectrometry-based omics technologies are of increasing relevance for drug discovery. In order to map drug treatments to biological pathways for mode of action (MoA) deconvolution, they rely on the determination of multiple testing corrected significant molecular events. The main contributors to statistical significance are the effect size and the variation of a given molecule during the drug treatment. While the former is largely determined by the biological system and the impact of the drug, the latter reflects the robustness of the experimental workflow. The impact of sample preparation and instrumentation on variation are well characterized, but the impact of cell culture conditions on variation of omics readouts is less explored, specifically related to subculture conditions during cell expansion. Here, we provide evidence that memory effects from subculture directly influence the variation of multilevel omics readouts, including oxylipins and fatty acids, proteins and phosphorylated sites, and phenotypic parameters, e.g. cell motility. When comparing drug treatments after homogeneous and heterogeneous subculture, the coefficients of variation (CVs) of controls increased from 1% to 3% for oxylipins and fatty acids, 11% to 23% for proteomics, 24% to 53% for phosphoproteomics and 6% to 14% for cell motility, respectively. The number of identifications remained comparable and the fold-change distributions were also largely similar after both subculture conditions. Therefore, the decrease in CVs seems to be the main contributor to the increased number of significant molecular events in drug treatments after homogeneous subculture. This in turn increased information content in the obtained perturbation network, based on proteomics and phosphoproteomics data, from 58 to 321 causal conjectures. Perturbation networks after homogeneous and heterogeneous subculture allowed for delineating the established MoA of arsenic trioxide in detail, including enhanced kinase signaling and reduced MYC-related gene expression, induction of heat shock proteins and cell cycle disruption. However, only the perturbation network after homogeneous subculture enabled the discovery of unprecedented drug effects, including down-regulation and quadruple dephosphorylation of the transcription factor RB1, among others. In summary, memory effects from subculture have broad relevance for MoA testing in drug discovery since this impact clearly manifests in supernatants, whole cell lysates and phenotypic properties and its control enables a considerably enhanced MoA deconvolution, facilitating discovery efforts.

Project description:Microcystin-LR (MCLR) exposure has been associated with development of hepatocellular carcinoma (HCC). Many of the carcinogenic mechanisms for MCLR have been attributed to the induction of cell survival and proliferation through altered protein phosphorylation pathways by inhibition of protein phosphatases 1 (PP1) and PP2A. The current study determined MCLR effects on the phosphoproteome in human HepaRG cells. Differentiated HepaRG cells were treated with either vehicle or MCLR followed by phosphoproteomic analysis and Western blotting of MAPK-activated proteins. MCLR decreased cell viability at 24 h at doses as low as 0.03 µM. MCLR also caused a dose-dependent increase in phosphorylation of signaling and stress kinases. The number of decreased phosphosites by 0.1 µM MCLR was similar between the 2 h (212) and 24 h (154) timepoints. In contrast, a greater number of phosphosites were increased at 24 h (567) versus the 2 h timepoint (136), indicating the hyperphosphorylation state caused by MCLR-mediated inhibition of PPs is time-dependent. A kinase perturbation analysis predicted that MCLR exposure at both 2 h and 24 h increased the function of aurora kinase B (AURKB), checkpoint kinase 1 (CHEK1), and serum and glucocorticoid-regulated kinase 1 (SGK1). STRING database analysis of the phosphosites altered by MCLR exposure revealed pathways associated with cell proliferation and survival, including ribosomal protein S6 kinase (RSK), and vascular endothelial growth factor receptor (VEGFR2)-mediated vascular permeability. In addition, several cancer-related KEGG pathways were enriched at both 2 h and 24 h timepoints, and multiple cancer-related disease-gene associations were identified at the 24 h timepoint. Many of the kinases and pathways described above play crucial roles in the development of HCC by affecting processes such as invasion and metastasis. Overall, our data indicate that MCLR-mediated changes in protein phosphorylation involve biological pathways related to carcinogenesis that may contribute to the development of HCC.