MuFGPS: enhancing liquid–liquid phase separation protein prediction through multi-level features and ensemble learning

Liquid–liquid phase separation (LLPS) is a key mechanism driving the assembly of membrane-less organelles and is increasingly recognized for its involvement in essential cellular functions and various diseases. However, existing computational approaches largely rely on sequence-level descriptors and often fail to explicitly incorporate structural topology information, limiting their ability to capture the complex determinants of LLPS behavior. Accurate identification of LLPS-capable proteins remains challenging due to their sequence diversity and complex structural determinants. Here, we present MuFGPS (Multi-level Feature Graph-based Predictor for Phase-Separating proteins), a predictive framework integrating sequence-derived physicochemical features, Define Secondary Structure of Proteins-annotated secondary structures, and graph-based structural embeddings from AlphaFold residue contact maps via a multi-head Graph Attention Network. Class imbalance is addressed using Synthetic Minority Oversampling Technique (SMOTE), and classification is performed through a stacking ensemble of Random Forest, XGBoost, and LightGBM. Benchmarks against six representative methods demonstrate that MuFGPS achieves superior performance across all metrics, with notable gains in F1-score and matthews correlation coefficient (MCC). Ablation analyses confirm the synergistic contributions of structural features and ensemble learning to accuracy and robustness. MuFGPS offers a scalable and high-accuracy framework for proteome-wide LLPS protein prediction.