Bioinformatics for Proteomics 2D Gels

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Bioinformatics for Proteomics 2D Gels

• By Andrew Garrow,
• University of Leeds

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Myself

• PhD Bioinformatics for a proteomics approach to
  understanding the Schistosome tegument.
• Construction of a proteomics database
• 2D Gels
• Mass spectrometry
• Data analysis

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Lecture Layout

• Introduction to 2D gel electrophoresis
• 2D gel databases
• 2D gel analysis

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Proteomics

• Analysis by direct measurement of proteins in
  terms of their presence and relative abundance.

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Why study Proteomics?

• To better understand protein expression and
  formation
• Transcriptional control
• Post-transcriptional control e.g. alternate
  splicing, RNA editing
• Translational and degradation control,
  translational frameshifting
• gt200 known PTM e.g. phosphorylation,
  glycolysation, lipid attachment, peptide cleavage
• No proteins gt No genes

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2D Gels

• Used for the separation of proteins within a
  sample.
• Dependant upon protein molecular weight and pI.
• Can be used for to resolve gt1,800 spots (Choe and
  Lee, 2000).

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2D Gels
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The Problem

• 2D Gels can routinely be used to separate gt1000
  spots, yet cells express 1000s-10000s of
  proteins.
• Approaches to improve protein coverage
• Separation on the basis on differential
  compartmentalisation/solubilisation
• Narrow range IPG strips for focusing on
  particular pI ranges.

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Zooming

• Use narrow range IPG strips to focus on
  particular pI ranges.

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2D Gel Procedure

• Three day process
• Day 1 Rehydration phase
• Day 2 Isoelectric focusing (IEF)
• Day 3 Second dimension

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Staining
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Gel Analysis

• Gel images digitally captured using a
  charged-couple device (CCD) camera or scanner
• Analysed by specialised software
• Phoretix 2D Advanced (www.phoretix.com)
• PDQuest (www.proteomeworks.bio-rad.com)
• 2D Elite (http//www.imsupport.com/)
• Melanie (www.expasy.ch/melanie)

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Gel Analysis

• Software features
• Spot detection
• Spot quantification
• Noise reduction
• Gel comparison by warping
• Linkage with robots

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Robot Spot Cutter
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Post Gel analysis

• Robotic spot picking
• Protein/spot digestion e.g. with trypsin
• Mass spectrometry (MS)
• MS data analysis
• Data repository

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Data Repository

• Database a collection of data records either in
  a single file or in multiple files.
• Database management system (DBMS) a software
  suite including a database, the utilities
  required to organize it, search, update, maintain
  data security and control access.
• Databases flat file, relational, object
  orientated.

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2D Gel Databases
   

•  

www.expasy.ch - Swiss-2DPAGE http//www.anl.gov/B
IO/PMG/ - Mouse liver, human breast cell lines,
pyrococcus. Argonne Protein Mapping Group.
  http//www.harefield.nthames.nhs.uk/nhli/protei
n/index.html - HSC-2DPAGE, Heart Science Centre,
Harefield Hospital http//oto.wustl.edu/thc/peri
-gels.htm - Washington Univ. Inner Ear Protein
Database http//ca.expasy.org/ch2d/2d-index.html
- World 2DPAGE, Index of 2D gel databases
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Federated 2D PAGE database

• Described by Appel et al (1996)
• Aimed to tackle (then) emerging problems with 2D
  Gel databases
• non-uniformity of data-encoding conventions
• robustness
• consistency
• commitment of groups to maintain the databases
  and data quality

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Federated 2D PAGE database

• Rules
• Rule 1 Individual entries in the database must
  be accessible by a keyword search. Other methods
  are possible but not required.
• Rule 2 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.
• Rule 3 A main index has to be supplied that
  provides a means of querying all databases
  through one unique query point. Currently, the
  main index is the SWISS-PROT database.
• Rule 4 Individual protein entries must be
  available through clickable images.
• Rule 5 2DE analysis software designed for use
  with federated databases, must be able to access
  individual entries in any federated 2DE
  databases.
• http//ca.expasy.org/ch2d/fed-rules.html

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Swiss 2DPAGE

• Established in 1993
• Maintained by the Central Clinical Chemistry
  Laboratory of the Geneva University Hospital and
  the Swiss Institute of Bioinformatics.
• Entries highly annotated -
• containing textual data on proteins including
  
• mapping procedure
• physiological and pathological information,
• experimental data (isoelectric point, molecular
  weight, amino acid composition, peptide masses)
• bibliographical references.

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Swiss 2DPAGE

• Entries are linked to images showing the
  experimentally determined and theoretical protein
  locations.
• Cross-references are provided to other federated
  2D-PAGE database entries, Medline and SWISS-PROT
• Search via - clickable images
• - keywords

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Make2DDB

• Software package provided by ExPASY
• Allows for production of a 2DPAGE database on
  users server.
• Database created which is queryable via
  description, accession or spot clicking.
• Provides links to Swiss-Prot.

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Make2DDB databases

• http//semele.anu.edu.au/2d/2d.html -
• ANU 2D-PAGE, Australian National University
  2D-PAGE database
• http//babbage.csc.ucm.es/2d/2d.html -
• COMPLUYEAST 2DPAGE, Saccharomyces cerevisae
  2D-PAGE database at Universidad complutense
  Madrid, Spain
• http//www.gram.au.dk/ -
• PHCI-2DPAGE, Parasite host cell interaction
  2D-PAGE interaction database.
• http//www.bio-mol.unisi.it/2d/2d.html -
• Sienna 2D PAGE
• A sample of 2D-PAGE databases created with
  make2ddb.

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2D Gel Databases

• 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.

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Proteomics Database Schema

• What should it encompass?
• Proteomics methods (e.g. protein sample prep,
  electrophesis buffers, staining techniques,
  digestion for MS etc).
• Results from each stage of the experiment (e.g.
  gel images, MS data).
• Parameters used for MS data analysis/statistical
  results
• All stored in strict format.
• Note MIAME and MAGE-ML

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Database querying

• Interact via web interface using Perl/CGI
• Clickable gel images
• Text querying for keywords, gel/spot name,
  author, sequence etc.
• XML used for data exchange

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Proteomics Database Schema
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Introduction to databases

• Flat file simplest database type, an ordered
  collection of data entries, analogous to how
  files would be stored in a filing cabinet.
• Relational more sophisticated, storing data in
  inter-related tables. Allow for flexible querying
  using Structured Query Language (SQL).
• Object Orientated database consistent with
  object orientated principles, allowing for
  storage of complex datatypes (i.e. multimedia)
  and querying beyond that defined by a rigidly
  defined query language.

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DBMS choice

• A flat file database would contain many
  redundancies in storing complex data types.
• An object-oriented database could intrinsically
  store complex data types e.g. large images,
  however, a relational database could contain
  links to images stored elsewhere.
• SQL would provide a fast and easy way of querying
  and updating the database.
• A relational database would provide a platform,
  easily expandable to accommodate additional forms
  of data.

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Future

• Standard database schema for proteomics and
  mark-up language for data exchange.
• Improved spot detection, quantification and gel
  warping algorithms.
• Improved sample preparation techniques.
• More automation (linkage of robots!).
• Protein array technologies.

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References

• Appel RD, et al 1993 - SWISS-2DPAGE a database
  of two-dimensional gel electrophoresis images.
  Electrophoresis, 14, 1232-1238.
• Appel RD, Bairoch A, Sanchez JC, Vargas JR, Golaz
  O, Pasquali C and Hochstrasser DF, 1996
  Federated two-dimensional electrophoresis
  database a simple means of publishing
  two-dimensional electrophoresis data,
  Electrophoresis, 17, 540-546.
• Bjellqvist B, Ek K, Righetti PG, Gianazza E, Gorg
  A, Westermeier R, Postel W., 1982 Isoelectric
  focusing in immobilised pH gradients principle,
  methodology and applications, J.Biochem.Biophys.Me
  thods, 6, 317-339.
• Brazma A, et al. 2001 Minimum information about
  a microarray experiment (MIAME)-towards standards
  for microarray data, Nat. genetics, 29, 365-71.
• Hoogland C, Baujard, Sanchez JC Hochstrasser DF
  and Appel RD, 1997 Make2ddb a simple package
  to set up a two-diensional electrophoresis
  database for the world wide web, Electrophoresis,
  18, 2755-2758.
• O'Farrell, 1975 - High resolution two-dimensional
  electrophoresis of proteins., J.Biol.Chem., 25,
  250, 4007-21.