Abstract
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.
Highlights
Phosphorylation is the most studied post-translational modification (PTM) due to its central role in cellular regulation
To associate phosphorylated sites of unknown functions to potential cellular functions, we developed an algorithm to apply to shotgun phosphoproteomics data
random walk on the heterogeneous network (RWHN) and over-representation analysis (ORA) were applied as done previously. We found that both RWHN and ORA could assign these sites to the function “negative regulation of cell growth” (Figure 5A)
Summary
Phosphorylation is the most studied post-translational modification (PTM) due to its central role in cellular regulation. It is thought to be the principal PTM in the human proteome and an essential mediator of protein−protein interactions (PPIs) and protein functions.[1] Transient changes occur at regulated phosphorylation sites, of which there may be multiple on each protein. Functional analysis of phosphoproteomics datasets is typically based on gene-centric enrichment of Gene Ontology (GO) terms or involvement in known pathways.[4] this approach disregards information captured by phosphoproteomics data on changes at specific phosphorylated sites, by limiting the analysis to the protein level. Analyses are hampered by the lack of phosphorylation site-specific functional annotations
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