EBI is an Outstation of the European Molecular Biology Laboratory.

UniProt and Complete proteomes

Sandra Orchard

Importance of reference protein sequence databases

• Completeness and minimal redundancyA 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 nomenclatureDatabases 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

Computational analysis and biological interpretation of shotgun proteomic data requires a reference protein sequence database

Summary of some protein sequence databases used in proteomicsDatabase Description Species

UniProtKB Expertly 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

UniRef100 Assembled from UniProtKB, Ensembl and RefSeq; merges 100% identical sequences; stable identifiers

Many

Ensembl Predictions 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 genomesExpanded Ensembl Metazoa, Plants and Fungi, Protists, Bacteria and Archaea

RefSeq NCBI 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

IPI Good balance between degree of redundancy and completeness; references to primary data sources; attempts to maintain stable identifiers; Assembled from UniProtKB, RefSeq and Ensembl

7 eukaryotic genomes

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

Protein sequences: UniProt databases

4

IPI Closure• IPI was launched in 2001 to cover the gaps in gene

predictions between different databases

• It is an integrated database which clusters protein sequences from different databases (e.g. UniProt, Ensembl and Refseq) to provide non-redundant complete data sets for human, mouse, rat, zebrafish, arabidopsis, chicken and cow.

• Collaboration efforts between Ensembl, Refseq and UniProt to improve gene prediction quality coverage for many of the most-studied genomes.

Collaboration projects between database resources• CCDS project, a collaboration between Ensembl, NCBI,

UCSC and UniProt, aims to provide a standard set of gene predictions for the human and mouse genomes• Considerable communication effort between curators from

different groups is on-going• Ensembl and UniProt collaboration to cover the gaps in

gene predictions in UniProtKB (one sequence for each protein coding transcript in Ensembl)• Ensembl high quality gene/transcript models (quality checks

remove gene models with erroneous structures or supported by dubious evidence – e.g. cDNA fragments with short/wrongly annotated ORF)

• UniProtKB high quality protein sequences

Complete proteome data set for IPI species

• Complete proteome data sets for IPI species are based on existing UniProtKB sequences supplemented by high quality predictions imported from Ensembl

• Includes the UniProtKB/Swiss-Prot manual annotated protein sequences supplemented by protein sequences in UniProtKB/TrEMBL from high quality predictions cross-referenced or imported from Ensembl

Complete proteome data sets for IPI species in UniProtKB• Ensembl sequences have now been incorporated for all

the species represented in IPI: human, mouse, rat , zebrafish, chicken and cow.

• Also for species such as dog, pig, C.elegans, Drosophila melanogaster and Saccharomyces cerevisiae.

• Complete proteome keyword by release 2011_07 of 28th June

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

• Will work in other species of interest

Manual annotation of the human proteome(UniProtKB/Swiss-Prot) – May 2010

• A draft of the complete human proteome has been available in UniProtKB/Swiss-Prot since 2008

• Manually annotated representation of 20,252 protein coding genes with over 35,000 protein sequences - an additional 41,000 UniProtKB/TrEMBL form the complete proteome set

• Approximately 63,000 single amino acid polymorphisms (SAPs), mostly disease-linked

• 80,000 post-translational modifications (PTMs)• Close collaboration with NCBI, Ensembl, Sanger Institute

and UCSC to provide the authoritative set to the user community

Finding a complete proteome in UniProtKB

Complete Proteomes

Selecting a sequence set

UniProtKB/TrEMBL

Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to:

o Erroneous gene model predictionso Sequence errors (Frame shifts)o Polymorphismso Alternative start siteso Isoforms

Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.

Sequence Sequence features

Ontologies

ReferencesNomenclature

Splice variants

Annotations

UniProtKB

Manual annotation of UniProtKB/Swiss-Prot

Master headline

Sequence curation, stable identifiers, versioning and archiving

For example – erroneous gene model predictions, frameshifts….

..premature stop codons, read-throughs, erroneous initiator methionines…..

Master headline

Splice variants

Master headline

Domain annotation

Binding sites

Master headline

Identification of amino acid variants

..and of PTMs

Statistics of Protein Modifications

Number of different modifications produced from each of the encoded amino acids, as currently annotated in the RESID Database of Protein Modifications (523 total).

15 Jul 2010

D E N Q S T C M H K R G A P V L I F Y W fM U O0

20406080

100120140160

Number of Different Modifications

Statistics of Protein Modifications

Number of modifications produced from each of the encoded amino acids in all the proteins currently

annotated in the UniProt Knowledgebase.

15 Jul 2010

D E N Q S T C M H K R G A P V L I F Y W fM U O0

50000

100000

150000

200000

250000

300000

350000

400000

Number of Annotated Modifications in UniProt

Understanding PTMs – additional resources

High quality PTM annotation required for peptide identification – must take additional weight of any PTM into account

RESID (www.ebi.ac.uk/resid) - collection of annotations and structures for protein modifications and cross-links including pre-, co-, and post-translational modifications. - provides systematic and alternate names, atomic formulas and masses, enzymatic activities that generate the modifications, keywords, literature citations, Gene Ontology (GO) cross-references, protein sequence database feature table annotations, structure diagrams, and molecular models.

Master headline

Understanding PTMs – additional resources

Requirements for MS1. Both specific (O-phospho-L-serine) and ambiguous

(phosphorylation) chemical terms are needed to describe modifications at different levels of experimental resolution.

2. Must include protein cross-linking modifications.3. Must include artificial modifications4. Must include masses and mass differences5. Must provide for sequential modifications on the same

residue, and for neutral losses

Master headline

Understanding PTMs – additional resources

PSI-MOD - community standard ontology that reconciles descriptions of protein residue modifications across multiple resources

(UniProt, RESID, UniMOD, DeltaMass)

Master headline

www.ebi.ac.uk/ontology-lookup/browse.do?ontName=MOD

Master headline

Protein nomenclature

Master headline

Master headline

Controlled vocabularies used whenever possible…

Annotation - >30 defined fields

Master headline

..and also imported from external resources

Binary interactions taken from the IntAct database

Interactors of human p53

Master headline

Controlled vocabulary usage increasing – for example from the Gene Ontology

Annotation for human Rhodopsin

1 Evidence at protein levelThere 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)

2 Evidence at transcript levelThe 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.3 Inferred from homologyThe existence of a protein is likely because orthologs exist in closely related species

4 Predicted5 Uncertain

Sequence evidence Type of evidence that supports the existence of a protein

UniProtKB/TrEMBL

Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to:

o Erroneous gene model predictionso Sequence errors (Frame shifts)o Polymorphismso Alternative start siteso Isoforms

Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.

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

Master headline

Master headline

Searching UniProt – Simple Search

• Text-based searching• Logical operators ‘&’ (and), ‘|’

Master headline

Searching UniProt – Advanced Search

Master headline

Searching UniProt – Search Results

Each linked to the UniProt entry

Master headline

Searching UniProt – Search Results

Master headline

Searching UniProt – Search Results

Master headline

Searching UniProt – Blast Search

Master headline

Searching UniProt – Blast Search

Master headline

Searching UniProt – Blast Results

Alignment with query sequence

Master headline

Searching UniProt – Blast Results

Contributing to UniProtKB