1. EBI is an Outstation of the European Molecular Biology Laboratory. UniProtKB Sandra Orchard

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 Stability and 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 DatabaseDescriptionSpecies UniProtKBExpertly curated section (UniProtKB/Swiss-Prot) and computer- annotated section (UniProtKB/TrEMBL); minimum level of redundancy; high level of integration with other databases; stable identifiers; diversity of sources including large scale genomics, small scale cloning and sequencing, protein sequencing, PDB, predicted sequences from Ensembl and RefSeq Many UniRef100Assembled from UniProtKB, Ensembl and RefSeq; merges 100% identical sequences; stable identifiers Many EnsemblPredictions using automated genome annotation pipeline; explicitly linked to nucleotide and protein sequences; stable reference; merge their annotations with Vega annotations at transcript level; extensive quality checks to remove erroneous gene models ; high level of integration with other databases Over 50 Eukaryotic genomes Ensembl Genomes: Metazoa, Plants and Fungi, Protists, Bacteria and Archaea RefSeqNCBI creates from existing data; ongoing curation; non-redundant; explicitly linked nucleotide and protein sequences; stable reference; high level of integration with other databases Limited to fully sequenced organisms Entrez protein (NCBInr)Assembled from GenBank and RefSeq coding sequence translations and UniProt KB ; annotations extracted from source curated databases; high degree of sequence redundancy Many 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 Master headline UniProt Knowledgebase: 2 sections 1. UniProtKB/Swiss-Prot Non-redundant, high- quality manual annotation - reviewed 2. UniProtKB/TrEMBL Redundant, automatically annotated - unreviewed www.uniprot.org

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

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

7. Master headline Splice variants

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

9. Master headline Domain annotation Binding sites

10. Master headline Protein nomenclature

11. Master headline

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

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

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

15. 1Evidence 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) 2Evidence 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. 3Inferred from homology The existence of a protein is likely because orthologs exist in closely related species 4 Predicted 5Uncertain 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. Master headline Searching UniProt – Simple Search Text-based searching Logical operators ‘&’ (and), ‘|’

18. Master headline Searching UniProt – Advanced Search

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

20. Master headline Searching UniProt – Search Results

21. Master headline Searching UniProt – Search Results

22. Master headline Searching UniProt – Blast Search

23. Master headline Searching UniProt – Blast Search

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

25. Master headline Searching UniProt – Blast Results

26. UniProtKB/TrEMBL Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to: o Erroneous gene model predictions o Sequence errors (Frame shifts) o Polymorphisms o Alternative start sites o 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. Master headline 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 Automatic Annotation

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

30. Master headline InterPro

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 Fasta files by FTP One file per species containing canonical + isoform sequences

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