“proteomics & bioinformatics”
DESCRIPTION
“Proteomics & Bioinformatics”. MBI, Master's Degree Program in Helsinki, Finland. Lecture 4. 10 May, 2007. Sophia Kossida , BRF, Academy of Athens, Greece Esa Pit känen , Univeristy of Helsinki, Finland Juho Rousu , University of Helsinki, Finland. Proteomics and biology /Applications. - PowerPoint PPT PresentationTRANSCRIPT
“Proteomics & Bioinformatics”
MBI, Master's Degree Program in Helsinki, Finland
10 May, 2007
Sophia Kossida, BRF, Academy of Athens, Greece
Esa Pitkänen, Univeristy of Helsinki, Finland
Juho Rousu, University of Helsinki, Finland
Lecture 4
Proteome Mining
Identifying as many as possible of the proteins in your sample
Protein Expression Profiling
Identification of proteins in a particular sample as a function of a particular state of the organism or cell
Functional proteomics
Post-translational modifications
Identifying how and where the proteins are modified
Protein-protein interactions Protein-network mappingDetermining how the proteins interact with each other in living systems
Structural Proteomics
Protein quantitation or differential analysis
Proteomics and biology /Applications
Databases and tools
Melanie
Identification
Quantification
General workflow of proteomics analysis
External data sourcestaxonomy, ontologies, bibliography…
Applications Systems biology (pathways, interactions..) biomarker-discovery, drug targets
Proteins/peptides
2D gel image aquisition and storage
MALDI, MS/MS Store peak lists and all meta data
Digestion and/or separation
PMF
MS/MS
DIGE
LC-MS & Tags
Sequence data bases:EMBL Nucleotide Sequence Database GenBank UniProtKB/Swiss-Prot & TrEMBL Ensemble EST database PIR
Identification
Quantification
General workflow of proteomics analysis
Proteins/peptidesDigestion and/or separation
MALDI, MS/MS
2D Page data bases
Swiss 2D PAGE, Gelbank, Cornelia, WordPAGE
Make 2D
Imaging tools:Melanie, PDQuest ProgenesisDelta 2D
Storing/ organising:Proteincsape
MSight
KEGG PDB DIPOMIMReactomePROSITPfamSPINBONDSTRINGAmiGODavidPubMedMEDLINE
MascotSequestAldentePopitamPhenyxFindModProfoundPepFragMS-FitOMSSASearch XLinksTagIdent
General workflow of proteomics analysis
Proteins/peptidesDigestion and/or separation
2D Page data bases
Make 2D
Imaging Softwares:The ability to compare two gels (images) and then identify differently expressed spots
•Melanie•PDQuest•Progenesis•Delta 2D
Proteinscape –platform for storing, organizing dataMSight -representation of mass spectra along with data from the separation
2D gel databases:Data integration on the webImage data and textual information
•Swiss 2D PAGE •Gelbank •Cornelia•WordPAGE
2D Gel Databases
Swiss-2DPAGE www.expasy.ch
GelBank http://www.gelscape.ualberta.ca:8080/htm/gdbIndex.html
Cornea 2D-PAGE http://www.cornea-proteomics.com/
World 2DPAGE, Index of 2D gel databaseshttp://ca.expasy.org/ch2d/2d-index.html
Swiss 2D PAGE viewer
Gel bank
Cornea
It runs on most UNIX-based operating systems (Linux, Solaris/SunOS, IRIX). Being continuously developed, the tool is evolving in concert with the current Proteomics Standards Initiative of the Human Proteome Organization (HUPO).
Data can be marked to be public, as well as fully or partially private.
An administration Web interface, highly secured, makes external data integration, data export, data privacy control, database publication and versions' control a very easy task to perform.
A software package to create, convert, publish, interconnect and keep up to date 2DE-databases. Provided by ExPASY
The database is queryable via description, accession or spot clicking.
Cross-references are provided to other federated 2D PAGE database entries, Medline and SWISS-PROT
Entries are linked to images showing the experimentally determined and theoretical protein locations.
Search via –clickable images, -keywords
Make 2D database
Federated database
Limitations of current databases:Do not contain strict/detailed descriptions of protocol (buffers, sample volume, staining techniques all important information for gel comparisons).Designed as 2D (and not proteomics) databases and therefore not readily expandable to incorporate other proteomics data e.g. MS, MDLC.Designed for reference gels, not on-going projects.
Robustness Consistency Maintenance of the databaseData quality
A collection of databases that are treated as one entity and viewed through a single user interface (pc.mag.com)
Guidelines for building a federated 2-DE database
http://ca.expasy.org/ch2d/fed-rules.html
Individual entries in the database must be accessible by a keyword search. Other methods are possible but not required.
The database must be linked to other databases by active hypertext cross-references, linking together all related databases. Database entries must be at least linked to the main index.
A main index has to be supplied that provides a means of querying all databases through one unique query point.
Individual protein entries must be available through clickable images.2DE analysis software designed for use with federated databases, must be able to access individual entries in any federated 2DE databases.
for a complete reference, see Appel et al., Electrophoresis
17, 1996, 540-546, 1996):
Image analysis software
ImageMaster2D/ Melanie
PDQuest (Bio-Rad, USA)
Progenesis (Nonlinear, UK)
Delta2D (Decodon, Germany)
Melanie
http://au.expasy.org/melanie/
Melanie
http://www.2d-gel-analysis.com/
PDQuest
http://www.bio-rad.com/
http://www.nonlinear.com/products/progenesis/
Progenesis
ProteinScape
• Hierarchy:
Project
Sample
Gel
Spots
MS Data
Search Events
Platform for storing, organizing, analyzing data generated during the proteomics workflow.
MSightSpecifically developed for the representation of mass spectra along with data from the separation
http://www.expasy.org/MSight
Sequence data bases:EMBL Nucleotide Sequence DatabaseGenBank UniProtKB/Swiss-Prot & TrEMBLEnsembleEST databasePIR
Identification
Quantification
General workflow of proteomics analysis
MALDI, MS/MS Store peak lists and all meta data
PMF
MS/MS
DIGE
LC-MS & Tags
Proteins/peptides
2D gel image aquisition and storage
Digestion and/or separation
EMBL Nucleotide Sequence Database
Collaboration between GenBank (USA) and DNA Database of Japan (DDBJ) and EBI.
New collected sequence data is exchanged, and each database is updated daily.
EBI
GenBank
Each entry includes a concise description of the sequence, the scientific name and the taxonomy of the source organism, and a table of features that identifies coding regions and other sites of biological significance, such as transcription units, sites of mutations or modifications and repeats.
Protein translations for coding regions are included in the feature table.
Bibliographic references are included along with a link to the Medline unique identifier for all published sequences.
Gen Bank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences.
http://www.psc.edu/general/software/packages/genbank/genbank.html
GenBank is available for searching at NCBI
Search GenBank
http://www.ncbi.nlm.nih.gov/Genbank/index.html
DDBJ
INSDC
UniProtUniversal Protein Resource
Joining the information contained in UniProtKB/Swiss-Prot, UniProteKB/TrEMBL and PIR.
It is comprised of three components
•UniProt Knowledge base (curated protein information, including function, classification, and cross-reference.
•UniProt Reference Clusters (combines closely related sequences into a single record to speed searches.)
•UniProt Archive (is a repository, reflecting the history of all protein sequences)
ExPASy Proteomics Server
Expert Protein Analysis System
Proteomics server of the Swiss Institute of Bioinformatics (SIB) is dedicated to the analysis of protein sequences and structures as well as 2D-PAGE.
http://ca.expasy.org/
http://www.isb-sib.ch/
UniProtKB/Swiss-Prot
The UniProt KB/Swiss-Prot Protein Knowledgebase is a annotated protein sequence database established in 1986. It is maintained collaboratively by the SIB (Swiss Institute of Bioinformatics) and the European Bioinformatics Institute (EBI)
http://ca.expasy.org/sprot/
Swiss Prot
TrEMBL
•Uni ProtKB/TrEMBL is a computer-annotated protein sequence database complementing the UniProtKB/Swiss-Prot Protein Knowledgebase.
•It contains the translations of all coding sequences (CDS) present in the EMBL/GenBank/DDBJ Nucleotide Sequence Databases and also protein sequences extracted from the literature or submitted to UniProtKB/Swiss-Prot.
•The database is enriched with automated classification and annotation.
PIR
http://pir.georgetown.edu/pirwww/
ESTdb
Expressed Sequence Tags, EST is a unique DNA sequence within a coding region of a gene that is useful for identifying full-length genes and serves as a landmark for mapping.
The dbEST is a division of GenBank that contains sequence data and other information on “singke-pass” cDNA sequences, from a number of organisms.
http://www.ncbi.nlm.nih.gov/dbEST/
Ensemble
http://www.ebi.ac.uk/ensembl/
Ensemble is a joint project between the EMBL-EBI and the Welcome Trust Sanger Institute that aims at developing a system that maintains automatic annotation of large eukaryotic genomes. Access to all the software and data is free and without constraints of any kind.
IPI- International Protein Index
Identification
Quantification
General workflow of proteomics analysis
MALDI, MS/MS Store peak lists and all meta data
PMF
MS/MS
DIGE
LC-MS & Tags
Proteins/peptides
2D gel image aquisition and storage
Digestion and/or separation
MascotSequestAldentePopitamPhenyxFindModProfoundPepFragMS-FitOMSSASearch XLinksTagIdent
Proteomics tools
http://restools.sdsc.edu/biotools/biotools19.html
http://ca.expasy.org/tools/
PROWL
Identification and Characterization Tools
Mascot (Matrix Science)
Aldente (ExPasy)
Profound (Rockefeller University)
MS-Fit (Prospector; UCSF)
Sequest
Mascot
OMSSA
X!Hunter
PMFdata MS/MS data
Identification and Characterization Tools
Popitam (ExPASy, SIB)
Phenyx –GeneBio, Swizerland)
PepFrag (Rockefeller University, USA)
SearchXLinks – (Caesar, Germany)
Popitam
Popitam is designed to characterize peptides withunexpected modification (e.g. post-translational modifications or mutations) by tandem mass spectrometry (ExPASy, SIB)
http://expasy.org/cgi-bin/popitam/help.pl
Popitam results
Phenyx
Phenyx is a software platform for the identification and characterization of proteins and peptides from mass spectrometry data.
Developed by GeneBio in collaboration with SIB
http://www.phenyx-ms.com/about/about_phenyx.html
PEPFRAG
http://prowl.rockefeller.edu/
Searches known protein sequences with peptide fragment mass information
SearchXLinks
http://www.searchxlinks.de/
Analysis of mass spectra of modified, cross-linked, and digested proteins, the amino acid of which is known
Identification and Characterization Tools
FindMod predicts potential protein post-translational modifications (PTM) and finds potential single amino acid substitutions in peptides.
FindPept identifies peptides that result from unspecific cleavage of proteins from experimental masses, taking into account artefactual chemical modifications, posttranslational modifications (PTM) and protease autolytic cleavage.
GlycoMod predicts possible oligosaccharide structures that occur on proteins from their experimentally determined masses.
http://au.expasy.org/tools/findmod/
AACompIdent achieves identification with amino acid composition
TagIdent identifies proteins with isoelectric point, pI, molecular weight, MW, and sequence tag generating a list of proteins close to a given pI and Mw. Multident achieves cross-species identification with multiple parameters (pI, Mw, sequence tag and peptide mass fingerprinting data)
Identification
Quantification
General workflow of proteomics analysis
MALDI, MS/MS Store peak lists and all meta data
PMF
MS/MS
DIGE
LC-MS & Tags
Proteins/peptides
2D gel image aquisition and storage
Digestion and/or separation
KEGG PDB DIPOMIMReactomePROSITPfamSPINBONDSTRINGAmiGODavidPubMedMEDLINE
KEGG
http://www.genome.jp/kegg/kegg2.html
KEGG: Kyoto Encyclopedia of Genes and Genomes
•Organism specific entry points:
-KEGG Organisms
•Subject specific entry points:
-DRUG, GLYCAN, REACTION, KAAS
KEGG
Manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks for metabolism, other cellular processes, and human diseases.
Functional hierarchies and binary relations of KEGG objects, including genes and proteins, compounds and reactions, drugs and diseases, and cells and organisms.
Gene catalogs of all complete genomes and some partial genomes with ortholog annotation (KO assignment), enabling KEGG PATHWAY mapping and BRITE mapping.
A composite database of chemical substances and reactions representing our knowledge on the chemical repertoire of biological systems and environments.
KEGG is a “biological systems” database integrating both molecular building block information and higher-level systematic information.
Search Pathway
Carbon fixation
Search “Pathway”
“Pathways” _motifs
Reactome
Reactome
PubMed
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed
David
http://david.abcc.ncifcrf.gov/home.jsp
Protein Data Bank
http://www.rcsb.org/pdb/home/home.do
Provides a variety of tools and resources for studying the structures of biological macromolecules and their relationships to sequence, function, and disease.
OMIM
This database is a catalog of human genes and genetic disorders.
The database contains textual information and references. It also contains links to MEDLINE and sequence records
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
Protein family classification
PROSITE (ExPASY)
Pfam (Sanger Institute)
SMART (EMBL)
PrositA Pseudo-Rotational Online Service and Interactive Tool
Proteins can be grouped on the basis of their sequences, into a limited number of families.
Some regions have been better conserved than others during evolution. These regions are generally important for the function of a protein and/or the maintenance of the three- dimensional structure.
By analyzing the constant and variable properties of such groups of similar sequences, it is possible to derive a signature for a protein family or domain, which distinguishes its members from all other unrelated proteins.
http://au.expasy.org/prosite/
ww
PROSIT
PROSIT
PROSIT
Pfam
Multiple sequence alignments and HMMs of protein domains and families, at Sanger Institute.
http://www.sanger.ac.uk/Software/Pfam/help/index.shtml
Browse interactions
http://smart.embl-heidelberg.de/
Structure data bases/interactions
STRING (EMBL)
BOND (Unleashed Informatics)
Cytoscape
DIP (UCLA)
iHOP
SPIN-PP (protein-protein interfaces in the PDB)
MIPS (Mammalian Protein-Protein Interaction
Database)
InterAct (protein interactions from literature curation)
STRING search results
STRING graphical
STRING_ new node
BONDBOND
http://bond.unleashedinformatics.com
The Biomolecular Object Network Databank
Cytoscape
Cytoscape is an open source bioinformatics software platform for visualizing molecular interactions with gene expression profiles and other state data.
Node label position can be controled by new GUI in VizMapper.
Cytoscape_ plugins
Plugins available for network and molecular profile analysis.
for example:
•Filter the network•Find active subnetworks/ pathway modules•Find clusters
A tool to determine which Gene Ontology (GO) categories are statistically over respresented in a set of genes or a subgraph of a biological network.
Database of Interacting Proteins
The DIP database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent set of protein-protein interactions.
http://dip.doe-mbi.ucla.edu/
http://www.ihop-net.org/UniPub/iHOP/
iHOP