OOPSIE (OOPS informatics extension).

Motivation: The RNA-bound proteome represents a dynamic and pleiotropic network of RNA-binding proteins (RBPs) that integrate diverse cellular pathways. RNA-binding dynamics profiling using cross-linking-based enrichment strategies including Orthogonal Organic Phase Separation (OOPS), generates complex peptide-level quantitative mass spectrometry datasets, yet dedicated computational workflows for standardized normalization and differential analysis remain limited. Results: OOPS relies on phase partitioning of RNA binding proteomes, upon UV cross-linking. As glycoproteins have been shown to co-partition independently of cross-linking due to their physicochemical properties, they provide a cross-link-independent reference population that can be leveraged for normalization of RNA-binding-dependent changes. We introduce OOPSIE (Orthogonal Organic Phase Separation Informatics Extension), an R-based script that provides a reproducible framework of peptide-level OOPS-derived data using protein-level normalization and differential analysis of RNA-binding capacity. We demonstrate its utility using OOPS-derived datasets generated from A549 lung adenocarcinoma cells. Availability and implementation: OOPSIE is implemented in R as a standalone workflow and is freely available at https://github.com/CambridgeCentreForProteomics/oopsie, together with documentation and example datasets.