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Proteomics

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Protein Sequences from Mastodon and Tyrannosaurus Rex Revealed by Mass Spectrometry. John M. Asara,Mary H. Schweitzer, Lisa M. Freimark,Matthew Phillips, Lewis C. ... – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
  • What is a proteome?
  • Proteome Characterization
  • Proteomic Projects
  • Use of the Proteome

Reading Ch 17
BIO520 Bioinformatics Jim Lund
2
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
3
Gene vs. Protein diversity
  • Human Genome 25,000 genes
  • Human Proteome 300,000 to 1,200,000 protein
    variants

4
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

5
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!

6
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.

7
Components of Proteomics
Protein Separation
Mass Spectroscopy
Bioinformatics
8
Protein separation
  • 2D-PAGE
  • Separate proteins based on size and charge
  • Types of 2D-PAGE gels
  • IEF/SDS
  • NEPHGE/SDS
  • HPLC

9
2D-PAGE
10
Detection Methods
  • Stains
  • Fluorescence, Coomassie blue, silver stain
  • 500 spots
  • Radiolabeling
  • Coupled detection
  • Mass spectrometry
  • MALDI/TOF
  • ESI
  • Trypsin digestion/MS

11
Instrumentation
  • 2D gels
  • Simple
  • MALDI-TOF and variants
  • 200,000, benchtop

12
2D Gel Results
  • SwissProt
  • www.expasy.ch/ch2d/
  • 2DWG Meta-database of 2D-gels
  • http//www-lecb.ncifcrf.gov/2dwgDB/

13
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
14
2D-PAGE -gt MALDI-MS-gtPMF
15
2D-PAGE -gt MS-MS-gtPMF
16
MS-MS
17
Principles of MALDI-TOF Mass Spectroscopy
18
MALDI-TOF Results
19
A vs B
20
Analytical Approach to Peptide Mass
Fingerprinting Effect of Mass Tolerance
Search m/z Mass Tolerance (Da) Hits
Database
21
Analytical Approach to Peptide Mass
Fingerprinting Effect of Multiple Peptide Masses
Search m/z Mass Tolerance
Hits Database
22
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!

23
Methanol m/z spectra
24
Peptide Mass Fingerprinting
25
Protein Sequences from Mastodon and Tyrannosaurus
Rex Revealed by Mass Spectrometry
26
Iterative search protocol used for database
searching with protein mixtures.
27
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

28
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

29
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

30
Protein identification approaches
31
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

32
Clinical Diagnostics Proteomics Protein Profiling
33
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
34
Genome-Scale Biochemical Assay
  • Protein arrays-biochemically active

35
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?
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