Using Multilayer Heterogeneous Networks to Infer Functions of Phosphorylated Sites

[Image: see text] Mass spectrometry-based quantitative phosphoproteomics has become an essential approach in the study of cellular processes such as signaling. Commonly used methods to analyze phosphoproteomics datasets depend on generic, gene-centric annotations such as Gene Ontology terms, which do not account for the function of a protein in a particular phosphorylation state. Analysis of phosphoproteomics data is hampered by a lack of phosphorylated site-specific annotations. We propose a method that combines shotgun phosphoproteomics data, protein–protein interactions, and functional annotations into a heterogeneous multilayer network. Phosphorylation sites are associated to potential functions using a random walk on the heterogeneous network (RWHN) algorithm. We validated our approach against a model of the MAPK/ERK pathway and functional annotations from PhosphoSitePlus and were able to associate differentially regulated sites on the same proteins to their previously described specific functions. We further tested the algorithm on three previously published datasets and were able to reproduce their experimentally validated conclusions and to associate phosphorylation sites with known functions based on their regulatory patterns. Our approach provides a refinement of commonly used analysis methods and accurately predicts context-specific functions for sites with similar phosphorylation profiles.