Proteomics

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Proteomics

• Global Proteomics Identify and catalog all
  possible proteins in an organism.
• Targeted Proteomics Identify and catalog a
  subset of proteins involved in a specific
  pathway, disease, or other specific facet you
  wish to study.
• Structural proteomics Determine the 3-D
  structure of all proteins.

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Examples of Proteomics

• Chang et al., 2000. Patterns of Protein
  Synthesis and Tolerance of Anoxia in Root Tips of
  Maize Seedlings Acclimated to a Low-Oxygen
  Environment, and Identification of Proteins by
  Mass Spectrometry. Plant Physiology. 122
  295317.
• Gallardo et al., 2002. Proteomics of Arabidopsis
  Seed Germination. A Comparative Study of
  Wild-Type and Gibberellin-Deficient Seeds. Plant
  Physiology. 129 823837.
• Abraham J.K. Koo and John B. Ohlrogge. 2002.
  The Predicted Candidates of Arabidopsis Plastid
  Inner Envelope Membrane Proteins and Their
  Expression Profiles. Plant Physiology.
  130823-836. October 2002 issue
• My Research Identification of Phosphopeptides
  in Wheat that may be involved in disease
  resistance.

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Current protocol
Limited to 1000 proteins
A severe limitation if the entire genome is not
sequenced.
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2-Dimensional Gel Electrophoresis
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4O2\4H
8hr O2
8O2\4A
4O2\4H\4A
4\4\13
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Maize Root Tip Proteins During Normoxia
MALDI-DE-TOF
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Peptide Mass Finger Print Spectrum Spot 41
b (N-Term) Y (C-Term)
Mascot Peptide Mass Fingerprint (Bioinformatics)
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Proteomics of Arabidopsis Seed Germination. A
Comparative Study of Wild-Type and
Gibberellin-Deficient Seeds.
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The Predicted Candidates of Arabidopsis Plastid
Inner Envelope Membrane Proteins and Their
Expression Profiles.

• Plastid proteins selected via computer
• Found proteins with NH2-terminal
  plastid-targeting peptides from all annotated
  open reading frames from Arabidopsis.
• Membrane spanning domains
• Subtract known thylakoid membrane proteins
• This is only possible because the entire genome
  is sequenced.

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Identification of Phosphopeptides in Wheat

• Phosphoproteins are involved in the regulation of
  many aspects of cell physiology, development and
  response to pathogens and pests.

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• MS cannot deal with a complex mixture
• 10 or less peptides to the MS every 50
  microseconds.
• Proteins separated on 2-D gels can not be
  delivered to mass spectrometers in a form ready
  for analysis.
• Signature Peptide Approach with GIST (Global
  Isotope Stable Tagging)

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Analytical scheme for relative quantification of
control and treated samples
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RPC of ? - casein without IMAC selection
phosphoprotein
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RPC of ? - casein after Ga(III)-IMAC selection
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MALDI-TOF MS b-Casein RPC
Linear Mode
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ESI-MS and MS/MS of b-casein peptide
ELEELNVPGEIVEpSLpSpSpSEESITR
.
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Conclusions

• Ga(III)-IMAC is selective for phosphopeptides
• Can identify phosphopeptides via IMAC/RPC/MALDI

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Response to Septoria tritici

• Septoria tritici Resistant Line
• Mock-inoculated and inoculated
• Tissue samples collected 0, 1, 3, 6, 12, 24, 48,
  96 hr post inoculation
• Same material used for Functional Genomics

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RPC without Ga(III)-IMAC selection
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RPC with Ga(III)-IMAC selection
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Range of up and down regulated peptides at 0 hr
Ratio
Peptide mass
RPC Fraction
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Range of up and down regulated peptides at 6 hr
10 - 2 fold change
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Unchanged
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Down Regulation
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Up Regulation
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Character in bond indicates the possible
phosphorylation sites  
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References

• Klaas J. van Wijk. 2001. Challenges and Prospects
  of Plant Proteomics. Plant Physiology. 126
  501-508.
• Proteomics in Functional Genomics Protein
  Structure Analysis. P. Jolles and H. Jornvall.
  Ed. 2000. Birkhauser Verlag, Boston and Berlin.
•  Abraham J.K. Koo and John B. Ohlrogge. 2002. The
  Predicted Candidates of Arabidopsis Plastid Inner
  Envelope Membrane Proteins and Their Expression
  Profiles Plant Physiology. 130 823-836
• Volker Kruft, Holger Eubel, Lothar Jänsch, Wolf
  Werhahn, and Hans-Peter Braun 2001. Proteomic
  Approach to Identify Novel Mitochondrial Proteins
  in Arabidopsis Plant Physiology. 127 1694-1710
•  Karine Gallardo, Claudette Job, Steven P.C.
  Groot, Magda Puype, Hans Demol, Joël
  Vandekerckhove, and Dominique Job 2001.
  Proteomics of Arabidopsis Seed Germination. A
  Comparative Study of Wild-Type and
  Gibberellin-Deficient Seeds Plant Physiology.
  129 823-837.