PhosphoSitePlus® (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330 000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1 000 000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The ‘Regulatory sites’ dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The ‘PTMVar’ dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25 000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites.

PhosphoSitePlus (http://www.phosphosite.org) is an open, comprehensive, manually curated and interactive resource for studying experimentally observed post-translational modifications, primarily of human and mouse proteins. It encompasses 1 30 000 non-redundant modification sites, primarily phosphorylation, ubiquitinylation and acetylation. The interface is designed for clarity and ease of navigation. From the home page, users can launch simple or complex searches and browse high-throughput data sets by disease, tissue or cell line. Searches can be restricted by specific treatments, protein types, domains, cellular components, disease, cell types, cell lines, tissue and sequences or motifs. A few clicks of the mouse will take users to substrate pages or protein pages with sites, sequences, domain diagrams and molecular visualization of side-chains known to be modified; to site pages with information about how the modified site relates to the functions of specific proteins and cellular processes and to curated information pages summarizing the details from one record. PyMOL and Chimera scripts that colorize reactive groups on residues that are modified can be downloaded. Features designed to facilitate proteomic analyses include downloads of modification sites, kinase–substrate data sets, sequence logo generators, a Cytoscape plugin and BioPAX download to enable pathway visualization of the kinase–substrate interactions in PhosphoSitePlus®.

BioPAX (Biological Pathway Exchange) is a standard language to represent biological pathways at the molecular and cellular level. Its major use is to facilitate the exchange of pathway data (http://www.biopax.org). Pathway data captures our understanding of biological processes, but its rapid growth necessitates development of databases and computational tools to aid interpretation. However, the current fragmentation of pathway information across many databases with incompatible formats presents barriers to its effective use. BioPAX solves this problem by making pathway data substantially easier to collect, index, interpret and share. BioPAX can represent metabolic and signaling pathways, molecular and genetic interactions and gene regulation networks. BioPAX was created through a community process. Through BioPAX, millions of interactions organized into thousands of pathways across many organisms, from a growing number of sources, are available. Thus, large amounts of pathway data are available in a computable form to support visualization, analysis and biological discovery.

PhosphoSite is a curated, web-based bioinformatics resource dedicated to physiologic sites of protein phosphorylation in human and mouse. PhosphoSite is populated with information derived from published literature as well as high-throughput discovery programs. PhosphoSite provides information about the phosphorylated residue and its surrounding sequence, orthologous sites in other species, location of the site within known domains and motifs, and relevant literature references. Links are also provided to a number of external resources for protein sequences, structure, post-translational modifications and signaling pathways, as well as sources of phospho-specific antibodies and probes. As the amount of information in the underlying knowledgebase expands, users will be able to systematically search for the kinases, phosphatases, ligands, treatments, and receptors that have been shown to regulate the phosphorylation status of the sites, and pathways in which the phosphorylation sites function. As it develops into a comprehensive resource of known in vivo phosphorylation sites, we expect that PhosphoSite will be a valuable tool for researchers seeking to understand the role of intracellular signaling pathways in a wide variety of biological processes.

In BriefPathway analysis of PTM data sets is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases. We have developed a PTM signatures database (PTMsigDB) providing curated phosphorylation signatures of kinases, perturbations and signaling pathways to enable site-specific PTM signature enrichment analysis (PTM-SEA). Application of PTM-SEA to phosphoproteomes of several cell lines perturbed with growth factors, cell cycle inhibitors, or a specific PI3K inhibitor demonstrated the potential of our site centric approach to study dysregulated pathways in cancers. Graphical Abstract Highlights• Database of PTM site-specific phosphorylation signatures of kinases, perturbations and signaling pathways (PTMsigDB).• PTM signature enrichment analysis (PTM-SEA) outperformed gene-centric analysis in detection of EGF induced phospho signaling events.• PI3K perturbation signatures were readily detected in PI3Ka inhibited human breast cancer cells.• PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.