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3 US HUPO: Bioinformatics for Proteomics 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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Protein Sequence Databases for ProteomicsThe good, the bad & the ugly
US HUPO: Bioinformatics for ProteomicsNathan Edwards – March 12, 2006
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Protein Sequence Databases
• Link between mass spectra and proteins• A protein’s amino-acid sequence provides
a basis for interpreting• Enzymatic digestion• Separation protocols• Fragmentation
• 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
LiteratureFunction
• Biological process, molecular function, cellular component
• Known and predictedFeatures
• Polymorphism, Isoforms, PTMs, Domains
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Database types
Curated• Swiss-Prot• PIR• RefSeq NP
Translated• TrEMBL• RefSeq XP, ZP
Omnibus• NCBI’s nr• MSDB• IPI
Other• PDB• HPRD• EST• Genomic
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Human Sequences
• Number of Human Genes is believed to be between 20,000 and 25,000
PIR ~ 10,500
SwissProt ~ 12,000
RefSeq ~ 28,000
IPI-HUMAN ~ 48,000
TrEMBL ~ 52,000
MSDB ~ 105,000
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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: GO:0003700;
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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
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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
• >• 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 protein’s organism…• …or the source of the biological sample
• The most reliable sequence annotation available
• Useful only to the extent that it is correct• NCBI’s taxonomy is widely used
• Provides a standard of sorts; Heirachical• Other databases don’t necessarily keep up
• Organism specific sequence databases are also 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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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?
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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 protein’s 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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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 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-01-01-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-00-00-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-00-03-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-01-03-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-00-04-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGKPRFIAVGYVDDTQFVRF
P13746-01-04-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGKPRFIAVGYVDDTQFVRF
P13746-00-05-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-01-05-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-01-00-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-00-02-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
P13746-01-02-00 MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRF
******************************************:*****************
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Swiss-Prot VarSplic Output
P13746-00-01-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYTQAASSDSAQ
P13746-01-01-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQ
P13746-00-00-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYTQAASSDSAQ
P13746-00-03-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYTQAASSDSAQ
P13746-01-03-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQ
P13746-00-04-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYTQAASSDSAQ
P13746-01-04-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQ
P13746-00-05-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYTQAASSDSAQ
P13746-01-05-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQ
P13746-01-00-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAASSDSAQ
P13746-00-02-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSS------DRKGGSYSQAASSDSAQ
P13746-01-02-00 SSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYSQAASSDSAQ
************************************* *******:*********
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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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Omnibus Database Redundancy Elimination
NCBI’s nr:Keeps all descriptions, separated by ^A
MSDB:Pecking order: PIR1-4, TrEMBL, GenBank, Swiss-Prot, NRL3D
IPI:All accessions, one description
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Description Elimination
• gi|12053249|emb|CAB66806.1| hypothetical protein [Homo sapiens]
• gi|46255828|gb|AAH68998.1| COMMD4 protein [Homo sapiens]
• gi|42632621|gb|AAS22242.1| COMMD4 [Homo sapiens]
• gi|21361661|ref|NP_060298.2| COMM domain containing 4 [Homo sapiens]
• gi|51316094|sp|Q9H0A8|COM4_HUMAN COMM domain containing protein 4
• gi|49065330|emb|CAG38483.1| COMMD4 [Homo sapiens]
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Description Elimination
• gi|2947219|gb|AAC39645.1| UDP-galactose 4' epimerase [Homo sapiens]
• gi|1119217|gb|AAB86498.1| UDP-galactose-4-epimerase [Homo sapiens]
• gi|14277913|pdb|1HZJ|B Chain B, Human Udp-Galactose 4-Epimerase: Accommodation Of Udp-N- Acetylglucosamine Within The Active Site
• gi|14277912|pdb|1HZJ|A Chain A, Human Udp-Galactose 4-Epimerase: Accommodation Of Udp-N- Acetylglucosamine Within The Active Site
• gi|2494659|sp|Q14376|GALE_HUMAN UDP-glucose 4-epimerase (Galactowaldenase) (UDP-galactose 4-epimerase)
• gi|1585500|prf||2201313AUDP galactose 4'-epimerase
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Description Elimination• gi|4261710|gb|AAD14010.1| chlordecone reductase [Homo sapiens]
• gi|2117443|pir||A57407 chlordecone reductase (EC 1.1.1.225) / 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.-) I [validated] – human
• gi|1839264|gb|AAB47003.1| HAKRa product/3 alpha-hydroxysteroid dehydrogenase homolog [human, liver, Peptide, 323 aa]
• gi|1705823|sp|P17516|AKC4_HUMAN Aldo-keto reductase family 1 member C4 (Chlordecone reductase) (CDR) (3-alpha-hydroxysteroid dehydrogenase) (3-alpha-HSD) (Dihydrodiol dehydrogenase 4) (DD4) (HAKRA)
• gi|7328948|dbj|BAA92885.1| dihydrodiol dehydrogenase 4 [Homo sapiens]
• gi|7328971|dbj|BAA92893.1|dihydrodiol dehydrogenase 4 [Homo sapiens]
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DNA to Protein Sequence
Derived from http://online.itp.ucsb.edu/online/infobio01/burge
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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 don’t know about what protein sequences result from a gene
• Recent revision of number of human genes suggest a bigger role for alternative splicing.
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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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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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