Protein Sequence Databases for Proteomics

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Protein Sequence Databases for Proteomics

• Nathan Edwards
• Center for Bioinformatics and Computational
  Biology
• University of Maryland, College Park

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Protein Sequence Databases

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

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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.

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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
• Derived Data
• Molecular weight, pI

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Database types
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SwissProt

• From ExPASy
• Expert Protein Analysis System
• Swiss Institute of Bioinformatics
• 180,000 protein sequence entries
• 9,000 species represented
• 12,000 Human proteins
• Highly curated
• Minimal redundancy
• Some restrictions on commercial use

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PIR

• Protein Information Resource
• Georgetown University Medical Center
• 280,000 protein sequence entries
• Highly curated
• Public domain resource
• 10,500 Human proteins
• Grew out of the Atlas of Protein Sequence and
  Structure (1965-1978) edited by Margaret Dayhoff.

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TrEMBL

• Translated EMBL nucleotide sequences
• European Molecular Biology Laboratory
• European Bioinformatics Institute (EBI)
• Computer annotated
• Only sequences absent from SwissProt
• 165,000 protein sequence entries
• 88,000 species
• 52,000 Human proteins

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RefSeq

• Reference Sequence
• From NCBI (National Center for Biotechnology
  Information), NLM, NIH
• Integrated genomic, transcript, and protein
  sequences.
• Varying levels of curation
• Reviewed, Validated, , Predicted,
• 1,350,000 protein sequence entries
• 44,000 reviewed
• 28,000 Human proteins

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RefSeq

• Particular focus on major research organisms
• Tightly integrated with genome projects.
• Curated entries NP accesssions
• Predicted entries XP accessions

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UniProt

• Universal Protein Resource
• Combination of
• Swiss-Prot
• TrEMBL
• PIR
• Knowledgebase is highly curated
• Similar sequence clusters are available
• 50, 90, 100 sequence similarity

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IPI

• International Protein Index
• From EBI
• For a specific species, combines
• UniProt, RefSeq, Ensembl
• Species specific databases
• 48,000 protein sequence entries
• Human, mouse, rat, zebra fish, arabidopsis

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NCBIs nr

• non-redundant
• Contains
• GenBank CDS translations
• RefSeq Proteins
• Protein Data Bank (PDB)
• SwissProt, TrEMBL, PIR
• Others
• Similar sequences suppressed
• 100 sequence similarity
• 1,800,000 protein sequence entries
• 33,000 species

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MSDB

• From the Imperial College (London)
• Combines
• PIR, TrEMBL, GenBank, SwissProt
• Distributed with Mascot
• so well integrated with Mascot

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Others

• HPRD
• Manually curated integration of literature
• PDB
• Focus on protein structure
• dbEST
• Part of GenBank - EST sequences
• Genome Sequences

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Human Sequences

• Number of Human Genes is believed to be between
  20,000 and 25,000

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DNA to Protein Sequence
Derived from http//online.itp.ucsb.edu/online/inf
obio01/burge
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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

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UCSC Genome Browser

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

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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

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Names / IDs

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

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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

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FASTA Format
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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.

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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 starting to
  become available.

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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

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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

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Controlled Vocabulary
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Controlled Vocabulary
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Controlled Vocabulary
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Controlled Vocabulary
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Controlled Vocabulary
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Ontology Structure

• NCBI Taxonomy
• Tree
• Gene Ontology (GO)
• Molecular function
• Biological process
• Cellular component
• Directed, Acyclic Graph (DAG)
• Unstructured labels
• Overlapping?

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Ontology Structure
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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

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Protein Families
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Protein Families
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Protein Families

• PROSITE, PFam, InterPro, PRINTS
• Gene Ontology
• Swiss-Prot keywords
• Differences
• Motif style, ontology structure, degree of manual
  curation
• Similarities
• Primarily sequence based, cross species

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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

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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

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Swiss-Prot Variant Annotations
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Swiss-Prot Variant Annotations
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Swiss-Prot Variant Annotations
Feature viewer
Variants
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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


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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


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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

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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

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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

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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

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Summary

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