UniProt and Complete proteomes
Sandra Orchard
EBI is an Outstation of the European Molecular Biology Laboratory.
Importance of reference protein sequence
databases
Computational analysis and biological interpretation of shotgun proteomic data
requires a reference protein sequence database
• 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
Summary of some protein sequence
databases used in proteomics
Database
Description
Species
UniProtKB
Expertly curated section (UniProtKB/Swiss-Prot) and computerannotated 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 genomes
Expanded 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 crossreferenced 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 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.
Manual annotation of UniProtKB/Swiss-Prot
Splice variants
Sequence
Sequence
features
UniProtKB
Ontologies
Annotations
Nomenclature
References
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
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Domain annotation
Binding sites
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Identification of amino acid variants
..and of PTMs
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Statistics of Protein Modifications
Number of Different Modifications
160
140
120
100
80
60
40
20
0
D E N Q
S
T
C M H K R G A
P
V
L
I
F
Y W fM U O
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
Statistics of Protein Modifications
Number of Annotated Modifications in UniProt
400000
350000
300000
250000
200000
150000
100000
50000
0
D E N Q S T C M H K R G A P V L
I
F Y W fM U O
Number of modifications produced from each of the
encoded amino acids in all the proteins currently
annotated in the UniProt Knowledgebase.
15 Jul 2010
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) crossreferences, protein sequence database feature table annotations,
structure diagrams, and molecular models.
Master headline
Understanding PTMs – additional resources
Requirements for MS
1. 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 modifications
4. Must include masses and mass differences
5. 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)
www.ebi.ac.uk/ontology-lookup/browse.do?ontName=MOD
Master headline
Protein nomenclature
Master headline
Master headline
Annotation - >30 defined fields
Controlled vocabularies used whenever possible…
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
Master headline
Sequence evidence
Type of evidence that supports the existence of a protein
1 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)
2 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.
3 Inferred from homology
The existence of a protein is likely because orthologs exist in closely
related species
4 Predicted
5 Uncertain
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.
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
Master headline
Searching UniProt – Simple Search
• Text-based searching
• Logical operators ‘&’ (and), ‘|’
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Searching UniProt – Advanced Search
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Searching UniProt – Search Results
Each linked to the UniProt entry
Master headline
Searching UniProt – Search Results
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Searching UniProt – Search Results
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Searching UniProt – Blast Search
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Searching UniProt – Blast Search
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Searching UniProt – Blast Results
Alignment with query sequence
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Searching UniProt – Blast Results
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Contributing to UniProtKB