PHOSIDA

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Link/URL:	www.phosida.de/
What:	Phosida - Phosphorylation Site Database
Who:	Florian Gnad Matthias Mann
Updates:	Genome Annotation Section
Upcoming events:	Inclusion of additional phosphoproteomes of various organisms
Web Services:
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About PHOSIDA

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PHOSIDA, the phosphorylation site database, integrates thousands of high-confidence in-vivo phosphosites identified by mass spectrometry-based proteomics in various species. It includes phosphoproteomes of organisms, which cover the entire phylogenetic tree representatively, ranging from bacteria such as Escherichia coli and Bacillus subtilis to eukaryotes such as yeast and human. It contains 81 phosphorylation sites on 79 E.coli proteins, for example.
E.coli results are described in Macek and Gnad et al..

Content

PHOSIDA contains phosphoproteomes of the following prokaryotes:

Template:Organism not found
Escherichia coli (79 phosphoproteins, 81 phosphosites)
Bacillus subtilis (78 phosphoproteins, 78 phosphosites)
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Using PHOSIDA

Browsing

The web user is able to search for any protein of interest within a specified organism. In the case of human, it is possible to search via IPI accession number, Swissprot identifier, gene symbol, protein name, peptide sequence, protein sequence or any substring that matches with the description of a given protein, for example.

Searching

For each protein, the user is presented with general features such as isoelectric point (pI), molecular weight, sequence, and description at the protein level in addition to corresponding GO accession numbers, which are directly hyperlinked to the detailed description of the annotation at the Gene Ontology website. In addition, a hyperlink to the Reactome database is provided, if the specified protein is annotated in Reactome (www.reactome.org).


Furthermore, annotated protein features such as active sites, binding sites, domains, and signal sequences are derived from the Swissprot database and illustrated for eukaryotic phosphoproteins, when clicking the ‘motifs/domains’ button. In the case of human, protein assignments were based on the IPI database, which is cross-referenced with the Swissprot database by PHOSIDA. Entries of both databases that correspond to the same proteins were aligned to derive the exact positions of these protein features. Already annotated phosphosites derived from Swissprot are transferred to the IPI sequences in the same way and listed when clicking the ‘sites (other sources)’ button. The aligned regions can be visualized via ‘check alignment’ buttons. Furthermore, corresponding literature is also provided.


Moreover, all phosphorylated sites that have been identified in our laboratory are displayed on the left panel. This presentment allows to check immediately whether phosphorylation sites occur within known domains or signaling sequence regions. Additionally, it becomes instantly clear whether a known biologically functional site such as binding sites is phosphorylated. In such cases, one can link the phosphorylation event to its functional impact. If the localization probability is lower than 0.995, it is enclosed in round brackets. When users click on any of the displayed phosphosites, the surrounding sequence and matching kinase motifs are shown.
Often, several phosphopeptides covering the same phosphosite are measured by mass spectrometry. These peptides are also listed along with their localization probabilities, Mascot scores, and MSQuant/PTM scores for each instance. In many cases, for example, the growth factor treatment, PHOSIDA contains quantitative and time-resolved data for the relative abundance of each phosphopeptide. These data are listed separately for peptides as a function of their sequences, degrees of phosphorylation, and further categories, such as experimental design or fraction. When moving the mouse over the ‘occurences’ button, protein entries sharing the same phosphopeptide of interest are listed along with the number of unique peptides that have been measured in one experimental assay. Each peptide is color coded according to the protein assignment: if the peptide sequence is marked in green, the selected protein has the maximum number of peptides in comparison to all other proteins that contain the same peptide. If the protein assignment is ambiguous because of another protein with the same number of identified peptides, the peptide is highlighted in blue. Red indicates that other proteins exceed the number of detected peptides in comparison to the selected phosphoprotein. Each feature of PHOSIDA is explained in the help menu, which is accessible via the ‘background’ menu or via clicking on the ‘question mark’ button at the page of interest.


The evolutionary section of PHOSIDA displays the results of the homology searches using an approximate phylogeny of all investigated species. Taxonomic divisions are displayed on-screen when the cursor is pointed at the phylogenetic tree. If the selected phosphoprotein is not homologous to any protein of a certain organism, that organism is highlighted in red. If the similarity between the sequence of the phosphoprotein and its homologous protein was the significantly best one in both directions, the given organism is highlighted in green. A higher similarity between the sequence of the homologous protein and another protein of the organism of the selected phosphoprotein suggests paralogy, which is indicated in blue. The full global alignment between the given phosphoprotein and the orthologous protein of a specific organism is shown, when the web user clicks on the organism button.


In addition, all phosphorylation sites that have been measured in our laboratory are listed on the right side. If users click on a phosphorylation site of interest, the conservation status of the selected phosphorylation site is indicated in red or green. Green points to conservation. For conserved phosphosites, the alignment of the surrounding sequence is displayed. With alignments between the phosphorylation site of interest and protein sequences from 70 organisms, PHOSIDA enables users to check the conservation of each site of each protein of interest. Furthermore, the conservation of matching motifs can immediately be checked. This enables the user to distinguish conserved motifs around the phosphosite from other motifs that also match the phosphosite but are not conserved and may thus be less likely to be functionally important or have appeared only recently in evolution.


The genome annotation section is accessible via the ‘notepad’ button located right next to the main ‘web book’ of MAPU 2.0 and PHOSIDA (Figure 8.4). At first, the user is required to select a certain species of interest. Then, the karyotype of the selected species is illustrated along with a link that connects to the EnsEMBL genome annotation webpage. If one clicks on one of the displayed chromosomes, a more detailed image of the chosen chromosome is shown along with general information such as length of the chromosome, number of known and predicted genes, quantity of occurring single nucleotide polymorphisms (SNPs), and number of gene transcripts. Besides these annotations derived from the EnsEMBL database, the number of gene transcripts that have been identified in our lab is posed.
We wished to use the proteomic data to annotate the genome. Thus, we extracted all measured peptides of each proteomic dataset and reassigned the given peptide sequences to gene transcripts that are annotated in the EnsEMBL database.
The genome annotation section is accessible via the ‘notepad’ button located right next to the main ‘web book’ of PHOSIDA. At first, the user is required to select a certain species of interest. Then, the karyotype of the selected species is illustrated along with a link that connects to the EnsEMBL genome annotation webpage. If one clicks on one of the displayed chromosomes, a more detailed image of the chosen chromosome is shown along with general information such as length of the chromosome, number of known and predicted genes, quantity of occurring single nucleotide polymorphisms (SNPs), and number of gene transcripts. Besides these annotations derived from the EnsEMBL database, the number of gene transcripts that have been identified in our lab is posed.
Furthermore, the chromosome is divided up into 93 bins: On the one hand, the number of transcripts that are annotated in EnsEMBL is displayed. If one clicks on one of the bin boxes, the EnsEMBL web page will pop up showing a detailed view of the selected chromosome region. On the other hand, the number of transcripts that have been detected in any of our uploaded projects is illustrated for each bin. Clicking on one of these bin buttons results into the listing of all identified gene transcripts along with the descriptions of the corresponding genes and exact localizations on the chromosome. Furthermore, a link is provided for each gene transcript that connects to the EnsEMBL homepage displaying the full annotation of the given transcript. In addition to the general annotation of the given gene transcript, the popped up EnsEMBL page will show all detected phosphorylation sites via the PHOSIDA DAS source.

Usage examples

Add links to additional pages describing success stories here.

p-values

p-values are the probability that a particular site is phosphorylated. If the p-value was greater than 75% (or 0.75/1), this was considered a high p-value score.

Technology

Web Services/API

The underlying database of PHOSIDA is based on mySQL. In addition, we used C#/ASP.NET to generate the dynamic web application.

References

[1] Gnad, F., Ren, S., Cox, J., Olsen, J. V., Macek, B., Oroshi, M., Mann, M., PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol. 2007, 8

[2] Macek, B., Gnad, F., Soufi, B., Kumar, C., Olsen, J., Mijakovic, I., Mann, M., Phosphoproteome analysis of E.coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr Phosphorylation. Mol. Cell. Proteomics 2007

[3] Macek, B., Mijakovic, I., Olsen, J. V., Gnad, F., Kumar, C., Jensen, P. R., Mann, M., The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol. Cell. Proteomics 2007, 6, 697–707

[4] Olsen, J., Blagoy, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., Mann, M., Global, in vivo, and site-specific phosphorylation dynamics in signalling networks. Cell 2006, 127, 635–648

External Links

PHOSIDA (Phosphorylation Site Database): http://www.phosida.com
MAPU (Max-Planck Unified Proteome Database): http://www.mapuproteome.com