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Protein Sequence Databases for Proteomics The good, the bad

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Title: Protein Sequence Databases for Proteomics The good, the bad


1
Protein Sequence Databases for ProteomicsThe
good, the bad the ugly
  • US HUPO Bioinformatics for Proteomics
  • Nathan Edwards March 12, 2006

2
Protein Sequence Databases
  • Link between mass spectra and proteins
  • A proteins amino-acid sequence provides a basis
    for interpreting
  • Enzymatic digestion
  • Separation protocols
  • Fragmentation
  • We must interpret database information as
    carefully as mass spectra.

3
More than sequence
  • Protein sequence databases provide much more than
    sequence
  • Names
  • Descriptions
  • Facts
  • Predictions
  • Links to other information sources
  • Protein databases provide a link to the current
    state of our understanding about a protein.

4
Much more than sequence
  • Names
  • Accession, Name, Description
  • Biological Source
  • Organism, Source, Taxonomy
  • Literature
  • Function
  • Biological process, molecular function, cellular
    component
  • Known and predicted
  • Features
  • Polymorphism, Isoforms, PTMs, Domains

5
Database types
6
Human Sequences
  • Number of Human Genes is believed to be between
    20,000 and 25,000

7
Accessions
  • Permanent labels
  • Short, machine readable
  • Enable precise communication
  • Typos render them unusable!
  • Each database uses a different format
  • Swiss-Prot P17947
  • Ensembl ENSG00000066336
  • PIR S60367 S60367
  • GO GO0003700

8
Names / IDs
  • Compact mnemonic labels
  • Not guaranteed permanent
  • Require careful curation
  • Conceptual objects
  • Swiss-Prot names changed last year!
  • ALBU_HUMAN
  • Serum Albumin
  • RT30_HUMAN
  • Mitochondrial 28S ribosomal protein S30
  • CP3A7_HUMAN
  • Cytochrome P450 3A7

9
Description / Name
  • Free text description
  • Human readable
  • Space limited
  • Hard for computers to interpret!
  • No standard nomenclature or format
  • Often abused.
  • COX7R_HUMAN
  • Cytochrome c oxidase subunit VIIa-related
    protein, mitochondrial Precursor

10
FASTA Format
11
FASTA Format
  • gt
  • Accession number
  • No uniform format
  • Multiple accessions separated by
  • One line of description
  • Usually pretty cryptic
  • Organism of sequence?
  • No uniform format
  • Official latin name not necessarily used
  • Amino-acid sequence in single-letter code
  • Usually spread over multiple lines.

12
Organism / Species / Taxonomy
  • The proteins organism
  • or the source of the biological sample
  • The most reliable sequence annotation available
  • Useful only to the extent that it is correct
  • NCBIs taxonomy is widely used
  • Provides a standard of sorts Heirachical
  • Other databases dont necessarily keep up
  • Organism specific sequence databases are also
    available.

13
Organism / Species / Taxonomy
  • Buffalo rat
  • Gunn rats
  • Norway rat
  • Rattus PC12 clone IS
  • Rattus norvegicus
  • Rattus norvegicus8
  • Rattus norwegicus
  • Rattus rattiscus
  • Rattus sp.
  • Rattus sp. strain Wistar
  • Sprague-Dawley rat
  • Wistar rats
  • brown rat
  • laboratory rat
  • rat
  • rats
  • zitter rats

14
Controlled Vocabulary
  • Middle ground between computers and people
  • Provides precision for concepts
  • Searching, sorting, browsing
  • Concept relationships
  • Vocabulary / Ontology must be established
  • Human curation
  • Link between concept and object
  • Manually curated
  • Automatic / Predicted

15
Controlled Vocabulary
16
Controlled Vocabulary
17
Controlled Vocabulary
18
Controlled Vocabulary
19
Controlled Vocabulary
20
Controlled Vocabulary
21
Ontology Structure
  • NCBI Taxonomy
  • Tree
  • Gene Ontology (GO)
  • Molecular function
  • Biological process
  • Cellular component
  • Directed, Acyclic Graph (DAG)
  • Unstructured labels
  • InterPro, Pfam, Swiss-Prot keywords
  • Overlapping?

22
Ontology Structure
23
Protein Families
  • Similar sequence implies similar function
  • Similar structure implies similar function
  • Common domains imply similar function
  • Bootstrap up from small sets of proteins with
    well understood characteristics
  • Usually a hybrid manual / automatic approach

24
Protein Families
25
Protein Families
26
Protein Families
  • PROSITE, PFam, InterPro, PRINTS
  • Swiss-Prot keywords
  • Differences
  • Motif style, ontology structure, degree of manual
    curation
  • Similarities
  • Primarily sequence based, cross species

27
Gene Ontology
  • Hierarchical
  • Molecular function
  • Biological process
  • Cellular component
  • Describes the vocabulary only!
  • Protein families provide GO association
  • Not necessarily any appropriate GO category.
  • Not necessarily in all three hierarchies.
  • Sometimes general categories are used because
    none of the specific categories are correct.

28
Protein Family / Gene Ontology
29
Sequence Variants
  • Protein sequence can vary due to
  • Polymorphism
  • Alternative splicing
  • Post-translational modification
  • Sequence databases typically do not capture all
    versions of a proteins sequence

30
Sequence Variants
  • Swiss-Prot a curated protein sequence database
    which strives to provide a high level of
    annotation (such as the description of the
    function of a protein, its domains structure,
    post-translational modifications, variants,
    etc.), a minimal level of redundancy and high
    level of integration with other databases
  • - Swiss-Prot web site front page

31
Sequence Variants
  • b) Minimal redundancy
  • Many sequence databases contain, for a given
    protein sequence, separate entries which
    correspond to different literature reports. In
    Swiss-Prot we try as much as possible to merge
    all these data so as to minimize the redundancy
    of the database. If conflicts exist between
    various sequencing reports, they are indicated in
    the feature table of the corresponding entry.
  • - Swiss-Prot User Manual, Section 1.1

32
Sequence Variants
  • IPI provides a top level guide to the main
    databases that describe the proteomes of higher
    eukaryotic organisms. IPI
  • 1. effectively maintains a database of cross
    references between the primary data sources
  • 2. provides minimally redundant yet maximally
    complete sets of proteins for featured species
    (one sequence per transcript)
  • 3. maintains stable identifiers (with
    incremental versioning) to allow the tracking of
    sequences in IPI between IPI releases.
  • - IPI web site front page

33
Sequence Variants
  • Swiss-Prot variants, isoforms and conflicts are
    retained as features
  • Script varsplic.pl can enumerate all sequence
    variants
  • Command-line options for full enumeration
  • -which full -varsplic -variant -conflict

34
Swiss-Prot Variant Annotations
35
Swiss-Prot Variant Annotations
36
Swiss-Prot Variant Annotations
Feature viewer
Variants
37
Swiss-Prot VarSplic Output
P13746-00-01-00 MAVMAPRTLLLLLSGALALTQTWAGSHSM
RYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF P13746-01-01-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFI
AVGYVDDTQFVRF P13746-00-00-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-00-03-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-01-03-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-00-04-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGKPRFIAVG
YVDDTQFVRF P13746-01-04-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGKPRFIAVG
YVDDTQFVRF P13746-00-05-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-01-05-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-01-00-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-00-02-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF P13746-01-02-00
MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVG
YVDDTQFVRF


38
Swiss-Prot VarSplic Output
P13746-00-01-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAA
VMWRRKSS------DRKGGSYTQAASSDSAQ P13746-01-01-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKG
GSYTQAASSDSAQ P13746-00-00-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSY
TQAASSDSAQ P13746-00-03-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSY
TQAASSDSAQ P13746-01-03-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSY
TQAASSDSAQ P13746-00-04-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSY
TQAASSDSAQ P13746-01-04-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSY
TQAASSDSAQ P13746-00-05-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSY
TQAASSDSAQ P13746-01-05-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSY
TQAASSDSAQ P13746-01-00-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSY
TQAASSDSAQ P13746-00-02-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSY
SQAASSDSAQ P13746-01-02-00
SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSY
SQAASSDSAQ


39
Omnibus Database Redundancy Elimination
  • Source databases often contain the same sequences
    with different descriptions
  • Omnibus databases keep one copy of the sequence,
    and
  • An arbitrary description, or
  • All descriptions, or
  • Particular description, based on source
    preference
  • Good definitions can be lost, including taxonomy

40
Omnibus Database Redundancy Elimination
  • NCBIs nr
  • Keeps all descriptions, separated by A
  • MSDB
  • Pecking order PIR1-4, TrEMBL, GenBank,
    Swiss-Prot, NRL3D
  • IPI
  • All accessions, one description

41
Description Elimination
  • gi12053249embCAB66806.1 hypothetical protein
    Homo sapiens
  • gi46255828gbAAH68998.1 COMMD4 protein Homo
    sapiens
  • gi42632621gbAAS22242.1 COMMD4 Homo
    sapiens
  • gi21361661refNP_060298.2 COMM domain
    containing 4 Homo sapiens
  • gi51316094spQ9H0A8COM4_HUMAN COMM domain
    containing protein 4
  • gi49065330embCAG38483.1 COMMD4 Homo
    sapiens

42
Description Elimination
  • gi2947219gbAAC39645.1 UDP-galactose 4'
    epimerase Homo sapiens
  • gi1119217gbAAB86498.1 UDP-galactose-4-epimera
    se Homo sapiens
  • gi14277913pdb1HZJB Chain B, Human
    Udp-Galactose 4-Epimerase Accommodation Of
    Udp-N- Acetylglucosamine Within The Active Site
  • gi14277912pdb1HZJA Chain A, Human
    Udp-Galactose 4-Epimerase Accommodation Of
    Udp-N- Acetylglucosamine Within The Active Site
  • gi2494659spQ14376GALE_HUMAN UDP-glucose
    4-epimerase (Galactowaldenase) (UDP-galactose
    4-epimerase)
  • gi1585500prf2201313AUDP galactose
    4'-epimerase

43
Description Elimination
  • gi4261710gbAAD14010.1 chlordecone reductase
    Homo sapiens
  • gi2117443pirA57407 chlordecone reductase (EC
    1.1.1.225) / 3alpha-hydroxysteroid dehydrogenase
    (EC 1.1.1.-) I validated human
  • gi1839264gbAAB47003.1 HAKRa
    product/3 alpha-hydroxysteroid dehydrogenase
    homolog human, liver, Peptide, 323 aa
  • gi1705823spP17516AKC4_HUMAN Aldo-keto
    reductase family 1 member C4 (Chlordecone reductas
    e) (CDR) (3-alpha-hydroxysteroid dehydrogenase)
    (3-alpha-HSD) (Dihydrodiol dehydrogenase 4) (DD4)
    (HAKRA)
  • gi7328948dbjBAA92885.1 dihydrodiol
    dehydrogenase 4 Homo sapiens
  • gi7328971dbjBAA92893.1dihydrodiol
    dehydrogenase 4 Homo sapiens

44
DNA to Protein Sequence
Derived from http//online.itp.ucsb.edu/online/inf
obio01/burge
45
Translated sequences
  • Gene models describe introns and exons
  • Start site?
  • Splice sites?
  • Alternative splicing?
  • ESTs provide limited evidence of transcription
    only
  • There is a lot we dont know about what protein
    sequences result from a gene
  • Recent revision of number of human genes suggest
    a bigger role for alternative splicing.

46
Genome Browsers
  • Link genomic, transcript, and protein sequence in
    a graphical manner
  • Genes, ESTs, SNPs, cross-species, etc.
  • UC Santa Cruz
  • http//genome.ucsc.edu
  • Ensembl
  • http//www.ensembl.org
  • NCBI Map View
  • http//www.ncbi.nlm.nih.gov/mapview

47
UCSC Genome Browser
  • Shows many sources of protein sequence evidence
    in a unified display
  • Can use EST accession as a location!

48
Summary
  • Protein sequence databases should be interpreted
    with as much care as mass spectra
  • Use controlled vocabularies
  • Understand the structure of ontologies
  • Take advantage of computational predictions
  • Look for sequence variants
  • Be careful with omnibus databases
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