Protein and Proteome Annotation

1
Protein and Proteome Annotation

• David Wishart
• University of Alberta
• Edmonton, AB
• david.wishart_at_ualberta.ca

2
Annotating 2D Gels
Trypsin Gel punch
p53
Trx
G6PDH
3
Is This Annotated?
p53
Information 1) pI 2) MW 3) name (abbr) 4)
accession 5) relative amnt
Trx
G6PDH
4
How About This?
Information 1) name (abbr) 2) accession 3)
relative amnt 4) coexpressors
5
Is This Annotated?
gtP12345 Sequence 1 GATTACAGATTACAGATTACAGATTACAGAT
TACAG ATTACAGATTACAGATTACAGATTACAGATTACAGA TTACAGA
TTACAGATTACAGATTACAGATTACAGAT TACAGATTAGAGATTACAGA
TTACAGATTACAGATT ACAGATTACAGATTACAGATTACAGATTACAGA
TTA CAGATTACAGATTACAGATTACAGATTACAGATTAC AGATTACAG
ATTACAGATTACAGATTACAGATTACA GATTACAGATTACAGATTACAG
ATTACAGATTACAG ATTACAGATTACAGATTACAGATTACAGATTACAG
A TTACAGATTACAGATTACAGATTACAGATTACAGAT
6
Protein Annotation

• Objective - identify and describe all the
  physico-chemical, functional and structural
  properties of a protein including its sequence,
  accession , mass, pI, absorptivity, solubility,
  active sites, binding sites, reactions,
  substrates, homologues, function, name(s),
  abundance, location, 2o structure, 3D structure,
  domains, pathways, interacting partners

7
Protein vs. Proteome Annotation

• Protein annotation is concerned with one or a
  small number (lt50) proteins from one or several
  types of organisms
• Proteome annotation is concerned with entire
  proteomes (gt2000 proteins) from a specific
  organism (or for all organisms) - need for speed

8
Different Levels of Annotation

• Sparse typical of many gel or microarray
  annotations, usually just includes name and
  accession number
• Moderate typical of many sequence databases or
  of experiments aimed at identifying protein
  complexes or ligands
• Detailed not typical (occasionally found in
  organism-specific databases)

9
Different Levels of Database Annotation

• GenBank (large of sequences, minimal
  annotation)
• PIR (large of sequences, slightly better
  annotation)
• SwissProt (small of sequences, even better
  annotation)
• Organsim-specific DB (very small of sequences,
  best annotation)

10
GenBank Annotation
11
PIR Annotation
12
Swiss-Prot Annotation
13
CCDB Annotation
14
CCDB Annotation
15
Ultimate Goal...

• To achieve the same level of protein/proteome
  annotation as found in CCDB for all
  genes/proteins from 2D GE data, from microarray
  data or for sequence databases in general

How?
16
Annotation Methods

• Annotation by homology (BLAST)
• requires a large, well annotated database of
  protein sequences
• Annotation by sequence composition
• simple statistical/mathematical methods
• Annotation by sequence features, profiles or
  motifs
• requires sophisticated sequence analysis tools

17
Annotation by Homology

• Statistically significant sequence matches
  identified by BLAST searches against GenBank
  (nr), SWISS-PROT, PIR, ProDom, BLOCKS, KEGG, WIT,
  Brenda, BIND
• Properties or annotation inferred by name,
  keywords, features, comments

Databases Are Key
18
Sequence Databases

• GenBank
• www.ncbi.nlm.nih.gov/
• EMBL/trEMBL
• www.ebi.ac.uk/trembl/
• DDBJ
• www.nig.ac.jp/
• PIR
• http//pir.georgetown.edu/
• SwissProt
• www.expasy.ch/sprot/
• UniProt
• http//www.pir.uniprot.org/

19
Structure Databases

• RCSB-PDB
• http//www.rcsb.org/pdb/
• MSD
• http//www.ebi.ac.uk/msd/index.html
• CATH
• http//www.biochem.ucl.ac.uk/bsm/cath/
• SCOP
• http//scop.mrc-lmb.cam.ac.uk/scop/

20
Expression Databases

• Swiss 2D Page
• http//ca.expasy.org/ch2d/
• SMD
• http//genome-www5.stanford.edu/MicroArray/SMD/
• ArrayExpress
• http//www.ebi.ac.uk/arrayexpress/
• Gene Expr. Omnibus
• http//www.ncbi.nlm.nih.gov/geo/

21
Metabolism Databases

• KEGG
• http//www.genome.ad.jp/kegg/metabolism.html
• Roche/Boeringer
• http//www.expasy.org/cgi-bin/search-biochem-index
  
• EcoCyc
• www.ecocyc.org/
• MetaCyc
• http//metacyc.org/

22
Interaction Databases

• BIND
• http//www.blueprint.org/bind/bind.php
• DIP
• http//dip.doe-mbi.ucla.edu/
• MINT
• http//mint.bio.uniroma2.it/mint/
• IntAct
• http//www.ebi.ac.uk/intact/index.html

23
Bibliographic Databases

• PubMed Medline
• http//www.ncbi.nlm.nih.gov/PubMed/
• Science Citation Index
• http//isi4.isiknowledge.com/portal.cgi
• Your Local eLibrary
• www.XXXX.ca
• Current Contents
• http//www.isinet.com/isi/

24
Annotation by HomologyAn Example

• 76 residue protein from Methanobacter
  thermoautotrophicum (newly sequenced)
• What does it do?
• MMKIQIYGTGCANCQMLEKNAREAVKELGIDAEFEKIKEMDQILEAGLTA
  LPGLAVDGELKIMGRVASKEEIKKILS

25
PSI BLAST
Select Database
26
PSI-BLAST
27
PSI-BLAST
28
PSI-BLAST
29
Conclusions

• Protein is a thioredoxin or glutaredoxin
  (function, family)
• Protein has thioredoxin fold (2o and 3D
  structure)
• Active site is from residues 11-14 (active site
  location)
• Protein is soluble, cytoplasmic (cellular
  location)

30
Annotation Methods

• Annotation by homology (BLAST)
• requires a large, well annotated database of
  protein sequences
• Annotation by sequence composition
• simple statistical/mathematical methods
• Annotation by sequence features, profiles or
  motifs
• requires sophisticated sequence analysis tools

31
Annotation by Composition

• Molecular Weight
• Isoelectric Point
• UV Absorptivity
• Hydrophobicity

32
Where To Go
33
Isoelectric Point

• The pH at which a protein has a net charge0
• Q S Ni/(1 10pH-pKi)

34
UV Absorptivity

• OD280 (5690 x W 1280 x Y)/MW x Conc.
• Conc. OD280 x MW/(5690 X W 1280 x Y)

OH
N
35
Hydrophobicity

• Indicates Solubility
• Indicates Stability
• Indicates Location (membrane or cytoplasm)
• Indicates Globularity or tendency to form
  spherical structure

36
Annotation Methods

• Annotation by homology (BLAST)
• requires a large, well annotated database of
  protein sequences
• Annotation by sequence composition
• simple statistical/mathematical methods
• Annotation by sequence features, profiles or
  motifs
• requires sophisticated sequence analysis tools

37
Where To Go
38
Sequence Feature Databases

• PROSITE - http//www.expasy.ch/
• BLOCKS - http//blocks.fhcrc.org/
• DOMO - http//www.infobiogen.fr/services/domo/
• PFAM - http//pfam.wustl.edu
• PRINTS - http//www.biochem.ucl.ac.uk/bsm/dbrowser
  /PRINTS
• SEQSITE - PepTool

39
What Can Be Predicted?

• O-Glycosylation Sites
• Phosphorylation Sites
• Protease Cut Sites
• Nuclear Targeting Sites
• Mitochondrial Targ Sites
• Chloroplast Targ Sites
• Signal Sequences
• Signal Sequence Cleav.
• Peroxisome Targ Sites

• ER Targeting Sites
• Transmembrane Sites
• Tyrosine Sulfation Sites
• GPInositol Anchor Sites
• PEST sites
• Coil-Coil Sites
• T-Cell/MHC Epitopes
• Protein Lifetime
• A whole lot more.

40
Cutting Edge Sequence Feature Servers

• Membrane Helix Prediction
• http//www.cbs.dtu.dk/services/TMHMM-2.0/
• T-Cell Epitope Prediction
• http//syfpeithi.bmi-heidelberg.com/scripts/MHCSer
  ver.dll/home.htm
• O-Glycosylation Prediction
• http//www.cbs.dtu.dk/services/NetOGlyc/
• Phosphorylation Prediction
• http//www.cbs.dtu.dk/services/NetPhos/
• Protein Localization Prediction
• http//psort.nibb.ac.jp/

41
Subcellular Localization
42
Subcellular Localization
http//www.cs.ualberta.ca/bioinfo/PA/Sub/
43
Proteome Analyst (SubCell)
44
2o Structure Prediction

• PredictProtein-PHD (72)
• http//cubic.bioc.columbia.edu/predictprotein/
• Jpred (73-75)
• http//www.compbio.dundee.ac.uk/www-jpred/submit.
  html
• SAM-T02 (75)
• http//www.cse.ucsc.edu/research/compbio/HMM-apps/
  T02-query.html
• PSIpred (77)
• http//bioinf.cs.ucl.ac.uk/psipred/psiform.html

45
Putting It All Together
Seq Motifs
Composition
Homology
46
Putting It All Together

• PEDANT
• http//pedant.gsf.de/
• GeneQuiz
• http//jura.ebi.ac.uk8765/ext-genequiz/
• Magpie
• http//magpie.ucalgary.ca/
• Proteome Analyst
• http//www.cs.ualberta.ca/bioinfo/PA/

47
(No Transcript)
48
Programs Used By Pedant

• HMMER
• PSORT
• PREDATOR
• COILS
• FGENESH
• pI
• PROSEARCH
• TargetP

• SAPS
• NCBI-BLAST
• SEG
• InterProScan
• SignalP
• TMHMM
• tRNAscan-SE
• GENSCAN

49
Databases Used By Pedant

• EMBL
• PIR-PSD
• SWISS-PROT
• Functional Cat
• PROSITE
• TrEMBL

• Blocks
• PDB
• SCOP
• COGs
• Pfam
• STRIDE

50
(No Transcript)
51
http//jura.ebi.ac.uk8765/gqsrv/submit
52
GeneQuiz Functions

• Amino acid biosynthesis
• Biosynthesis of cofactors, prosthetic
  groups, carriers
• Cell envelope
• Cellular processes
• Central intermediary metabolism
• Energy metabolism
• Fatty acid and phospholipid metabolism
• Other categories
• Purines, pyrimidines, nucleosides, and
  nucleotides
• Regulatory functions
• Replication
• Transcription
• Translation
• Transport and binding proteins
• Unknown

53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
56
(No Transcript)
57
Home Page
58
Proteome Analyst

• Uses PSI-BLAST, PSI-PRED and motif analysis tools
• Extracts keyword information from homologues and
  uses Naïve Bayes classifiers to infer function
• Combines sequence motif and sequence profile
  information to complete functional classification
• Supports custom classifier/ontology

59
BacMap

• Picking up where we left off with the CCDB
  (Google bacmap)
• Idea is to generate a visual atlas of all (not
  just Escherichia coli) bacterial chromosomes and
  plasmids but with links to extensive genome
  annotation
• Attempt to re-use annotation and graphing tools
  originally developed for the CCDB

60
BacMap
http//wishart.biology.ualberta.ca/BacMap/
61
BacMap
62
Text Search Tools
63
Sequence Search Tools
64
Bacterial Biography Card
65
Genome Statistics
66
Proteome Statistics
67
BacMap

• Each genome has a short description of the
  organism and sequence data
• Supports zoomable, hyperlinked, clickable map
  views of the genome
• Supports text search of gene names, protein names
  and synonyms
• Supports BLAST search and supplies genome-wide
  stats
• Currently going through major update

Stothard P, et al. BacMap an interactive picture
atlas of annotated bacterial genomes. Nucleic
Acids Res. 2005 Jan 133 Database IssueD317-20.
68
What if Your Organism or Genome isnt in BacMap?
http//wishart.biology.ualberta.ca/basys/
69
BASys

• Bacterial Annotation System
• A publicly available web server that performs
  automated annotation of bacterial genomes given
  only the gene sequence of a chromosome or plasmid
• Takes about 24 hrs for an average genome (4
  megabases)
• Output includes images and annotation text (about
  70 fields for each gene)

70
Typical BASys Result
71
Conclusion

• Genome annotation is the same as proteome
  annotation required after any gene sequencing
  and gene ID effort
• Can be done either manually or automatically
• Need for high throughput, automated pipelines
  to keep up with the volume of genome sequence
  data
• Area of active research and development with
  about ½ of all bioinformaticians working on some
  aspect of this process