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UniProtKB

Sandra Orchard. UniProtKB. Importance of reference protein sequence databases. Completeness and minimal redundancy A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs.

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UniProtKB

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  1. Sandra Orchard UniProtKB

  2. Importance of reference protein sequence databases • Completeness and minimal redundancy A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs. Low degree of redundancy for facilitating peptide assignments • Stabilityand consistency Stable identifiers and consistent nomenclature Databases are in constant change due to a substantial amount of work to improve their completeness and the quality of sequence annotation • High quality protein annotation Detailed information on protein function, biological processes, molecular interactions and pathways cross-referenced to external source

  3. Summary of protein sequence databases Updated from Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4,1419–1440l

  4. UniProtKB • UniProt Knowledgebase: • 2 sections • UniProtKB/Swiss-Prot Non-redundant, high-quality manual annotation - reviewed • UniProtKB/TrEMBL Redundant, automatically annotated - unreviewed www.uniprot.org Master headline

  5. Manual annotation of UniProtKB/Swiss-Prot Splice variants Sequence Sequence features UniProtKB Ontologies Annotations References Nomenclature

  6. Sequence curation, stable identifiers, versioning and archiving • For example – erroneous gene model predictions, frameshifts • …. ..premature stop codons, read-throughs, erroneous initiator methionines….. Master headline

  7. Splice variants Master headline

  8. Identification of amino acid variants ..and of PTMs … and also Master headline

  9. Domain annotation Binding sites Master headline

  10. Protein nomenclature Master headline

  11. Master headline

  12. Annotation - >30 defined fields Controlled vocabularies used whenever possible… Master headline

  13. ..and also imported from external resources Binary interactions taken from the IntAct database Interactors of human p53 Master headline

  14. Controlled vocabulary usage increasing – for example from the Gene Ontology Annotation for human Rhodopsin Master headline

  15. Evidence at protein level There is experimental evidence of the existence of a protein (e.g. Edman sequencing, MS, X-ray/NMR structure, good quality protein-protein interaction , detection by antibodies) Evidence at transcript level The existence of a protein has not been proven but there is expression data (e.g. existence of cDNAs, RT-PCR or Northern blots) that indicates the existence of a transcript. Inferred from homology The existence of a protein is likely because orthologs exist in closely related species 4 Predicted 5 Uncertain Sequence evidence Type of evidence that supports the existence of a protein

  16. Manual annotation of the human proteome(UniProtKB/Swiss-Prot) A draft of the complete human proteome has been available in UniProtKB/Swiss-Prot since 2008 Manually annotated representation of 20,231 protein coding genes with 36,865 protein sequences - an additional 33,243 UniProtKB/TrEMBL form the complete proteome set Approximately 67,600 single amino acid polymorphisms (SAPs), mostly disease-linked ~75,500 post-translational modifications (PTMs) Close collaboration with NCBI, Ensembl, Sanger Institute and UCSC to provide the authoritative set to the user community

  17. Text-based searching • Logical operators ‘&’ (and), ‘|’ Searching UniProt – Simple Search Master headline

  18. Searching UniProt – Advanced Search Master headline

  19. Each linked to the UniProt entry Searching UniProt – Search Results Master headline

  20. Searching UniProt – Search Results Master headline

  21. Searching UniProt – Search Results Master headline

  22. Searching UniProt – Blast Search Master headline

  23. Searching UniProt – Blast Search Master headline

  24. Alignment with query sequence Searching UniProt – Blast Results Master headline

  25. Searching UniProt – Blast Results Master headline

  26. UniProtKB/TrEMBL • Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to: • Erroneous gene model predictions • Sequence errors (Frame shifts) • Polymorphisms • Alternative start sites • Isoforms • Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.

  27. Why do we need predictive annotation tools?

  28. Automatic Annotation • Automated clean-up of annotation from original nucleotide sequence entry • Additional value added by using automatic annotation • Recognises common annotation belonging to a closely related family within UniProtKB/Swiss-Prot • Identifies all members of this family using pattern/motif/HMMs in InterPro • Transfers common annotation to related family members in TrEMBL Master headline

  29. ← Taxonomy ← Publication ← Name (non-standard) ← Sequence

  30. InterPro Master headline

  31. Master headline

  32. Finding a complete proteome in UniProtKB

  33. Complete Proteomes

  34. MS Proteomics • Require each sequence (inc isoforms) to be present in the dataset as an separate entity for search engines to access • For higher organisms, with isoforms, expanded set made available on ftp site • Fastafiles by FTP • One file per species containing canonical + isoform sequences

  35. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Master headline

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