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Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.
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http://dx.doi.org/10.1002/pmic.202300267 | DOI Listing |
Life Sci Alliance
November 2025
Immunoregulation Research Group, Max Planck Institute of Biochemistry, Martinsried, Germany
Amino acid (AA) detection is fundamental for cellular function, balancing translation demands, biochemical pathways, and signaling networks. Although the GCN2 and mTORC1 pathways are known to regulate AA sensing, the global cellular response to AA deprivation remains poorly understood, particularly in non-transformed cells, which may exhibit distinct adaptive strategies compared with cancer cells. Here, we employed murine pluripotent embryonic stem (ES) cells as a model system to dissect responses to AA stress.
View Article and Find Full Text PDFUnlabelled: Tyrosine kinases (TKs) are frequently mutated or overexpressed in cancer, and TK inhibitors (TKIs) are an important therapeutic modality against TK-driven cancers, but many patients show an underwhelming response to TKIs prescribed on the basis of tumor genotype. To find cell-intrinsic TK signaling patterns which might be predictive of poor response to TKI exposure, we used high-sensitivity multiplexed mass spectrometry to quantify endogenous levels of 1,222 phosphotyrosine (pY) sites across the proteomes of TK-driven human cancer cell lines with variable response to genotype-matched TKIs. In direct comparisons between TKI-tolerant and TKI-sensitive lines with a common driver TK, we found that TKI treatment was equally effective at blocking driver TK signaling, and higher basal activity of the driver TK did not always predict higher sensitivity to TKI.
View Article and Find Full Text PDFJ Proteome Res
September 2025
School of Life Sciences, Technical University of Munich, Emil Erlenmeyer Forum 5, Freising 85354, Germany.
Current applications of mass-spectrometry-based proteomics range from single-cell to body fluid analysis, each presenting very different demands regarding sensitivity or sample throughput. Additionally, the vast molecular complexity of proteomes and the massive dynamic range of protein concentrations in these biological systems require highly performant chromatographic separations in tandem with the high speed and sensitivity afforded by modern mass spectrometers. In this study, we focused on the chromatographic aspect and, more specifically, systematically evaluated proteome analysis performance across a wide range of chromatographic flow rates (0.
View Article and Find Full Text PDFDrug Metab Dispos
July 2025
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Division of Molecular Biosciences, University at Buffalo, Buffalo, New York. Electronic address:
Src family kinases, including the Lck/Yes-related novel protein kinase (LYN), have emerged as posttranslational modulators of various transporters involved in clinically relevant pharmacokinetic drug-drug interactions. LYN expression was recently detected in hepatocytes, and we hypothesized that LYN deficiency alters the phosphorylation status and activity of transporters involved in hepatic drug disposition. An untargeted phospho-proteomic screen in livers of mice revealed that LYN deficiency was associated with significantly reduced phosphorylation of the hepatic uptake organic anion transporting polypeptide Oatp1b2 transporter that recognizes a wide range of structurally diverse xenobiotics.
View Article and Find Full Text PDFSci Transl Med
September 2025
University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
is among the most frequently mutated oncogenes in cancer, and for decades, efforts at pharmacological blockade of its function in solid cancers have been unsuccessful. A notable advance in this endeavor is the recent development of small-molecule KRAS inhibitors, which enable direct targeting of the mutant oncoprotein. Here, we comprehensively evaluated the preclinical efficacy of BI-2493, a first-in-class allele-agnostic mutant-KRAS inhibitor (panKRASi), in pancreatic ductal adenocarcinoma (PDAC).
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