InstaNovo-P T47D cell phosphoproteomics

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.