Dataset Information

InstaNovo-P T47D cell phosphoproteomics

ABSTRACT: Phosphorylation, a vital post-translational modification (PTM), plays a central role in cellular signaling and disease mechanisms. Mass spectrometry-based phosphoproteomics is widely used for system-wide characterization of phosphorylation events. However, traditional methods struggle with accurate phosphosite localization, complex search spaces, and detecting sequences outside the reference database. Advances in de novo peptide sequencing offer opportunities to address these limitations, but have yet to be integrated and adapted for phosphoproteomics experiments. Here, we present InstaNovo-P, a phospho-specific version of our transformer-based InstaNovo model, fine-tuned on extensive phosphoproteomics datasets. InstaNovo-P significantly surpasses existing methods in phosphopeptide detection and phosphosite localization accuracy across multiple datasets, including complex experimental scenarios. Our model robustly identifies peptides with single and multiple phosphorylation sites, effectively localizing phosphorylation events on serine, threonine, and tyrosine residues. Experimental validation with FGFR2 signaling data further demonstrated that InstaNovo-P uncovers numerous phosphosites previously missed by traditional database searches, which align with critical biological processes, confirming the model’s capacity to yield valuable biological insights. InstaNovo-P adds value to phosphoproteomics experiments by effectively identifying biologically relevant phosphorylation events without prior information, providing a powerful analytical tool for the dissection of signaling pathways.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Konstantinos Kalogeropoulos

LAB HEAD: Chiara Francavilla

PROVIDER: PXD062859 | Pride | 2026-03-17

REPOSITORIES: Pride

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			Action	DRS
	20211118_Ex_CF_JH_T47D-FGFR2KO_F10_40min_STY_01_01.raw	Raw
	20211118_Ex_CF_JH_T47D-FGFR2KO_F10_40min_STY_01_02.raw	Raw
	20211118_Ex_CF_JH_T47D-FGFR2KO_F10_40min_STY_02_01.raw	Raw
	20211118_Ex_CF_JH_T47D-FGFR2KO_F10_40min_STY_02_02.raw	Raw
	20211118_Ex_CF_JH_T47D-FGFR2KO_F10_40min_STY_03_01.raw	Raw

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Project description:Dysregulated kinase activity drives oncogenic signalling, perturbs cellular homeostasis, and promotes tumour progression. Despite major success in targeting kinases therapeutically, the downstream consequences of kinase inhibition and the mechanisms underlying drug resistance remain incompletely understood. One of the most frequent oncogenic kinase mutations, BRAFV600E, constitutively activates the MAPK pathway and represents a major therapeutic target in melanoma and other cancers. However, the functional relevance of most phosphorylation events downstream of BRAF signalling is unknown, limiting mechanistic interpretation and rational therapeutic design. Here, we established a global, multi-omic model of BRAF inhibition response in BRAFV600E-mutant melanoma cells by integrating time-resolved phosphoproteomics, biophysical PTM-proteomics, transcriptomics, and thermal proteome profiling. Our ultradeep phosphoproteomic analysis revealed widespread phosphorylation changes upon Dabrafenib treatment, while biophysical phosphoproteomics uncovered phosphorylation events associated with altered solubility and subcellular localisation, indicative of biomolecular condensation and nuclear reorganisation. Integration of these modalities into a network-based mechanistic model enabled the prioritisation of functionally relevant phosphorylation sites and kinases. Experimental validation confirmed CDK9, CLK3, and TNIK as key regulators of BRAFV600E signalling and as candidate targets for combinatorial inhibition strategies capable of re-sensitising resistant melanoma cells in a synthetic lethal manner. The transcription factor ETV3 emerged from the network as a previously unrecognised effector of oncogenic BRAF signalling. Using phosphosite-specific biophysical data, imaging, and FRAP experiments, we demonstrated that ETV3 phosphorylation controls its DNA-binding kinetics. Functional assays combining ETV3 knockdown, metabolomics, and drug screening revealed that ETV3 modulates transcriptional and metabolic responses to BRAF inhibition, linking oncogenic signalling to metabolic rewiring. Together, this study provides a comprehensive systems-level framework that connects phosphorylation dynamics to protein function and cellular phenotype, highlights ETV3 as a novel signalling node, and illustrates how multi-omic, site-resolved network models can reveal actionable mechanisms of kinase-driven oncogenesis.

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InstaNovo-P T47D cell phosphoproteomics

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