Protein Sequence Databases

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Protein Sequence Databases

• Nathan Edwards
• Department of Biochemistry and Mol. Cell.
  Biology
• Georgetown University Medical Center

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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
Curated Swiss-Prot UniProt RefSeq NP Translated TrEMBL RefSeq XP, ZP
Omnibus NCBIs nr MSDB IPI Other PDB HPRD EST Genomic
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SwissProt

• From ExPASy
• Expert Protein Analysis System
• Swiss Institute of Bioinformatics
• 515,000 protein sequence entries
• 12,000 species represented
• 20,000 Human proteins
• Highly curated
• Minimal redundancy
• Part of UniProt Consortium

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TrEMBL

• Translated EMBL nucleotide sequences
• European Molecular Biology Laboratory
• European Bioinformatics Institute (EBI)
• Computer annotated
• Only sequences absent from SwissProt
• 10.5 M protein sequence entries
• 230,000 species
• 75,000 Human proteins
• Part of UniProt Consortium

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UniProt

• Universal Protein Resource
• Combination of sequences from
• Swiss-Prot
• TrEMBL
• Mixture of highly curated (Swiss-Prot) and
  computer annotation (TrEMBL)
• Similar sequence clusters are available
• 50, 90, 100 sequence similarity

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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,
• 9.7 M protein sequence entries
• 209,000 reviewed, 90,000 validated
• 39,000 Human proteins

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RefSeq

• Particular focus on major research organisms
• Tightly integrated with genome projects.
• Curated entries NP accessions
• Predicted entries XP accessions
• Others YP, ZP, AP

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IPI

• International Protein Index
• From EBI
• For a specific species, combines
• UniProt, RefSeq, Ensembl
• Species specific databases
• HInv-DB, VEGA, TAIR
• 87,000 (from 307,000 ) human protein sequence
  entries
• Human, mouse, rat, zebra fish, arabidopsis,
  chicken, cow

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MSDB

• From the Imperial College (London)
• Combines
• PIR, TrEMBL, GenBank, SwissProt
• Distributed with Mascot
• so well integrated with Mascot
• 3.2M protein sequence entries
• Similar sequences suppressed
• 100 sequence similarity
• Not updated since September 2006 (obsolete)

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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
• 10.5 M protein sequence entries

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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

SwissProt 20,000
RefSeq 39,000
TrEMBL 75,000
IPI-HUMAN 87,000
MSDB 130,000
nr 230,000
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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

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PeptideMapper Web Service
Im Feeling Lucky
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PeptideMapper Web Service
Im Feeling Lucky
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Unannotated Splice Isoform
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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
• 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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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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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
• Swiss-Prot keywords
• Differences
• Motif style, ontology structure, degree of manual
  curation
• Similarities
• Primarily sequence based, cross species

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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.

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Protein Family / Gene Ontology
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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 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

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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

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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

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Swiss-Prot Variant Annotations
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Swiss-Prot Variant Annotations
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Swiss-Prot Variant Annotations
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Peptides to Proteins
Nesvizhskii et al., Anal. Chem. 2003
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Peptides to Proteins
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Peptides to Proteins

• A peptide sequence may occur in many different
  protein sequences
• Variants, paralogues, protein families
• Separation, digestion and ionization is not well
  understood
• Proteins in sequence database are extremely
  non-random, and very dependent

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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
• Peptides to proteins not as simple as it seems
• Be careful with omnibus databases