Proteomics

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Proteomics

• What is a proteome?
• Proteome Characterization
• Proteomic Projects
• Use of the Proteome

Reading Ch 17
BIO520 Bioinformatics Jim Lund
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Protein Sequences from Mastodon and Tyrannosaurus
Rex Revealed by Mass SpectrometryJohn M.
Asara,Mary H. Schweitzer, Lisa M.
Freimark,Matthew Phillips, Lewis C.
CantleyScience 13 April 2007 Vol. 316. no.
5822, pp. 280 - 285
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Gene vs. Protein diversity

• Human Genome 25,000 genes
• Human Proteome 300,000 to 1,200,000 protein
  variants

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1 gene-gt many proteins

• Alternative splicing 60 of genes in the human
  genome, several splice forms are typical.
• International Human Genome Sequencing Consortium,
  Nature 2001
• Proteolytic cleavage
• Covalent modifications
• Phosphorylation, Acylation, Methylation,
  Glycosylation,Sulfation, Prenylation, lipid
  linkage and many more

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mRNA-protein Correlation

• YPD should have relevant data
• will yeast be typical?
• Electrophoresis 18533
• 23 proteins on 2D gels
• r0.48 for mRNAprotein
• Post-transcriptional and post translational
  regulation important!

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Branches of Proteomics

• Protein separation. Basic to all proteomic
  technologies are protein separation the
  separation of a complex mixture so that
  individual proteins are more easily processed
  with other techniques.
• Protein identification. Low-throughput sequencing
  through Edman degradation High throughput
  proteomic techniques based on mass spectrometry,
  commonly peptide mass fingerprinting on simpler
  instruments, or de novo sequencing with tandem
  mass spectrometry.
• Protein quantification. Gel-based methods such as
  differential staining of gels with fluorescent
  dyes (difference gel electrophoresis). Gel-free
  methods include various tagging or chemical
  modification methods, such as isotope-coded
  affinity tags (ICATs) or combined fractional
  diagnoal chromatography (COFRADIC). Mass spec
  methods are now giving quantification data.
• Protein sequence analysis. Bioinformatic branch,
  search databases for possible protein or peptide
  matches.
• Structural proteomics. High-throughput
  determination of protein structures in
  three-dimensional space using x-ray
  crystallography and NMR spectroscopy.
• Interaction proteomics. Investigation of protein
  interactions using IP then MS, 2-hydrid screens,
  protein chips.
• Protein modification. Almost all proteins are
  modified from their pure translated amino-acid
  sequence, so-called post-translational
  modification. Specialized methods have been
  developed to study phosporylation
  (phosphoproteomics) and glycosylation
  (glycoproteomics). MS methods are also used.

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Components of Proteomics
Protein Separation
Mass Spectroscopy
Bioinformatics
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Protein separation

• 2D-PAGE
• Separate proteins based on size and charge
• Types of 2D-PAGE gels
• IEF/SDS
• NEPHGE/SDS
• HPLC

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2D-PAGE
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Detection Methods

• Stains
• Fluorescence, Coomassie blue, silver stain
• 500 spots
• Radiolabeling
• Coupled detection
• Mass spectrometry
• MALDI/TOF
• ESI
• Trypsin digestion/MS

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Instrumentation

• 2D gels
• Simple
• MALDI-TOF and variants
• 200,000, benchtop

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2D Gel Results

• SwissProt
• www.expasy.ch/ch2d/
• 2DWG Meta-database of 2D-gels
• http//www-lecb.ncifcrf.gov/2dwgDB/

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Basic Proteomic Analysis Scheme
Separation
Protein Mixture
Individual Proteins
2D-SDS-PAGE
Spot Cutting
Digestion Trypsin
Mass Spectroscopy
Peptides
Peptide Mass
MALDI-TOF
Database Search
Protein Identification
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2D-PAGE -gt MALDI-MS-gtPMF
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2D-PAGE -gt MS-MS-gtPMF
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MS-MS
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Principles of MALDI-TOF Mass Spectroscopy
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MALDI-TOF Results
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A vs B
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Analytical Approach to Peptide Mass
Fingerprinting Effect of Mass Tolerance
Search m/z Mass Tolerance (Da) Hits
Database
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Analytical Approach to Peptide Mass
Fingerprinting Effect of Multiple Peptide Masses
Search m/z Mass Tolerance
Hits Database
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Peptide Mass Fingerprinting

• Find peptide fragments from MS spectra.
• Charge state deconvolution.
• Make peptide fragment list.
• Check versus list of all possible polypeptides.
• Need to have Protein database!

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Methanol m/z spectra
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Peptide Mass Fingerprinting
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Protein Sequences from Mastodon and Tyrannosaurus
Rex Revealed by Mass Spectrometry
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Iterative search protocol used for database
searching with protein mixtures.
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Peptide Search Programs

• Mascot
• http//www.matrixscience.com/
• SEQUEST
• http//fields.scripps.edu/sequest/ (Thermo
  Scientific)
• X!Tandem
• http//thegpm.org/
• Free and Open Source
• PeptIdent
• Uses Swiss-Prot protein database
• http//au.expasy.org/tools/peptident.html
• Free and Open Source
• ProteinProspector
• Searches user supplied protein database.
• http//falcon.ludwig.ucl.ac.uk/mshome3.2.htm
• Free and Open Source
• GFS
• Uses raw genome sequence.
• http//gfs.unc.edu/cgi-bin/WebObjects/GFSWeb
• Free and Open Source

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MS varieties

• Ionization Methods
• EI (Electron Impact)
• CI (Chemical Ionization)
• MALDI
• ESI
• Fast Atom Bombardment (FAB)
• Mass Analyzers
• Ion Trap
• Time-of-Flight
• Theoretically, no limitation for m/z maximum,
  high throughput
• Magnetic Sector
• High resolution, exact mass
• Ion Cyclotron Resonance (FTMS)
• Very high resolution, exact mass, perform ion
  chemistry
• Quadrupole
• Unit mass resolution, fast scan, low cost
• Technology is developing quickly!
• UK has a Proteomics Facility!
• http//www.rgs.uky.edu/ukmsf/proteomics.html

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Searching parameters

• Modifications (e.g. cysteine residues, etc.)
• Number of allowable missed cleavages
• Data properties monoisotopic/average, charge
  state, amino acid composition
• MS/MS data

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Protein identification approaches
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Limitations-2D gel, MS

• Protein preparation/electrophoresis
• hydrophobic proteins insoluble
• Sensitivity
• stains, 1 ng (1/10,000)
• MS (1 fmol, 40 pg for average)
• Protein modifications?unclear
• Data analysis/comparison
• Scimagix (www.scimagix.com)
• Nature of data
• quaternary info lost, localization lost

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Clinical Diagnostics Proteomics Protein Profiling
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Protein Arrays

• Many variants
• Diverse set of techniques
• Different targets, detection technologies
• Antibody Arrays (Hypromatrix)
• PepChip Arrays (Pepscan Systems)
• Active Protein Arrays (Protein One)

Overview of protein chipshttp//www.proteomicworl
d.org/ProteinArraysPage.html
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Genome-Scale Biochemical Assay

• Protein arrays-biochemically active

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Value of Proteome Data

• Contains info not in mRNA!
• mRNA ! protein
• Covalent modification of proteins critical to
  regulation, often with constant expression
• Association state of proteins critical
• How can we use this information?