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.

The functional landscape of the human phosphoproteome

“…For benchmarking purposes, we compared the identified phosphoproteome against the 221,236 human phosphosites reported by MS in the PSP database (January 2018) ( Figure 1e ) (Hornbeck et al 2019) . While 11.5% of the high-confidence sites in our study are supported by only 1 MS/MS evidence (mass spectrum), 55% of the PSP sites have this level of support.…”

“…Notably, an in-depth study of a single cell type identified over 50,000 phosphopeptides and suggested that 75% of the proteome may be phosphorylated (Sharma et al 2014) . Such studies have led to the identification of a large number of human phosphosites with over 200,000 currently aggregated in the PhosphoSitePlus (PSP) resource (Hornbeck et al 2019) .…”

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non‐Histone Substrates

“…To further develop the detection of possible novel substrates, the PhosphoSite Plus database (https://www.phosphosite.org/) was used, which allows to identify proteins with known methylation within the broadened SMYD2 sequence motif [LF]‐[K]. With this approach, 155 methylation sites in non‐histone proteins were identified which are methylated in cells at potential SMYD2 target sites (Table S2).…”

Protein Phosphorylation Dynamics: Unexplored Because of Current Methodological Limitations

“…These curated lists, which are perfectly annotated and searchable, represent a great advance for comparison with computational prediction based on the consensus linear sequences that specify each kinase. [ 25–36 ] The curated data are accessible at Cell Signaling Technology PhosphoSitePlus (http://www.phosphosite.org), phosphoPep (http://www.phosphopep.org/), phosphoGRID (https://phosphogrid.org/), Kinexus PhosphoNET (http://www.phosphonet.ca), and dbPAF (http://dbpaf.biocuckoo.org/). The last database resource reports 483 001 known phosphorylation sites in 54 148 proteins from Homo sapiens , Mus musculus , Rattus norvegicus , Drosophila melanogaster , Caenorhabditis elegans , Schizosaccharomyces pombe , and Saccharomyces cerevisiae .…”

“…Furthermore, the redundancy between these databases is a precious help for cross‐examination. Moreover, integrated datasets can form combinatorial resources which reports multiple high‐throughput analysis of phosphorylation from different public databases, such as Phospho.ELM, [ 26,27 ] dbPTM, [ 28 ] PHOSIDA, [ 29 ] PhosphoSitePlus, [ 30–32 ] PhosphoPep, [ 33 ] PhosphoGRID, [ 34 ] SysPTM, [ 35 ] HPRD, [ 36 ] and UniProt. [ 37 ] In parallel and for comparison, a large‐scale analysis of the linear sequence motif(s) for each known kinase was performed under the “Phosphorylome Project” using yeast proteome microarrays [ 38 ] : more than eighty purified yeast kinases or kinase complexes were assayed in the presence of 33 P‐γ‐ATP and more than 13 000 peptide substrates.…”