15 years of PhosphoSitePlus®: integrating post-translationally modified sites, disease variants and isoforms

Abstract

For 15 years the mission of PhosphoSitePlus® (PSP, https://www.phosphosite.org) has been to provide comprehensive information and tools for the study of mammalian post-translational modifications (PTMs). The number of unique PTMs in PSP is now more than 450 000 from over 22 000 articles and thousands of MS datasets. The most important areas of growth in PSP are in disease and isoform informatics. Germline mutations associated with inherited diseases and somatic cancer mutations have been added to the database and can now be viewed along with PTMs and associated quantitative information on novel 'lollipop' plots. These plots enable researchers to interactively visualize the overlap between disease variants and PTMs, and to identify mutations that may alter phenotypes by rewiring signaling networks. We are expanding the sequence space to include over 30 000 human and mouse isoforms to enable researchers to explore the important but understudied biology of isoforms. This represents a necessary expansion of sequence space to accommodate the growing precision and depth of coverage enabled by ongoing advances in mass spectrometry. Isoforms are aligned using a new algorithm. Exploring the worlds of PTMs and disease mutations in the entire isoform space will hopefully lead to new biomarkers, therapeutic targets, and insights into isoform biology.

Figure 1.

Visual simplicity of the new Homepage. Most of the text and features on the old Homepage have been moved or removed from the new Homepage. Two sets of searches, the ‘Protein or Substrate Search’, and the six searches in the ‘Advanced Search and Browse Section’ are still accessed directly from the Homepage. The ‘Site Statistics’ table has been moved to the ‘About’ menu, and the Motif and Sequence Logo tools have been moved to the ‘Tools’ menu.

Figure 2.

Find proteins in which missense mutations are associated with specific diseases or syndromes. (A) Disease query on the ‘Protein Sequence and Reference Search’ Page. (B) ‘Search Results’ lists proteins in which mutations are causally associated with the selected pathological state.Clicking on the protein selected in the ‘Search Results’ goes to (C) the ‘Protein Page’, where the ‘Protein Information’ section lists additional diseases or syndromes with associated links. The ‘Cancer’ tab opens a PSP page displaying statistics about the number of somatic mutations from TCGA observed for this protein.

Figure 3.

Redesigned Site Tables. Site Tables are found in one of the five tabs on the Protein Page. The sites and sequences from the selected species are displayed sequentially down the page. Clicking on a sequence opens a dropdown window displaying the sequences of homologous sites across all members of the protein group. Additional columns for other species or isoforms can be added by selecting the checkboxes above the site table. Effects of the PTM site on biological processes or the protein itself are shown by clicking on the icon displayed after the site sequence. Blue icons to the right of the number of HTP records lists site-specific antibodies available from Cell Signaling Technology.

Figure 4.

Visualizing PTM sites in lollipop plots. The lollipop plot illustrates phosphorylation (blue), acetylation (green), ubiquitylation (brown) and other (gray) PTM sites on human SHP-2. The horizontal axis displays protein residues and domains, while the vertical axis reflects the number of HTP and LTP papers reporting specified PTM sites of SHP-2. Zooming in (via buttons or mouse) allows the illustration of corresponding amino acids. The plot also shows details about PTM sites (here: phospho-Y63) triggered by hovering the mouse over lollipops.

Figure 5.

Adding disease mutations to lollipop plots. (A) The lollipop plot displays PTM sites and disease-associated SNPs (purple squares) on human SHP-2. The inclusion of disease-associated SNPs yields an additional purple vertical axis reflecting the minor allele frequency (MAF), if available. Hovering the mouse over squares yields the display of further details about the disease SNP. (B) Analogously, hotspot mutations from TCGA can be visualized as red squares. In this case the additional vertical axis (red) reflects the total number of tumors having the specified mutation.

Figure 6.

Multiple sequence alignments by (A) Clustal Ω and (B) Mirage (https://github.com/TravisWheelerLab/Mirage) of eight isoforms of SORBS2/ArgBP2 (O94875).