lecture 9. functional genomics at the protein level: proteomics
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Lecture 9. Functional Genomicsat the Protein Level: Proteomics
Functional Genomics: Development and Application of Genome-Wide Experimental Approaches to Assess Gene Function by making use of the information and reagents provided by Structural Genomics
Goals of Functional Genomics:1)DNA2)RNA3) Protein4) Whole organism5) Society
Lander, E. 1996. The New Genomics: Global Views of Biology. Science 274: 536-539.
Goals of Proteomicsa) monitoring the expression and modification state of all proteins in a cell; comparison of proteomesbetween cells
b i) systematic catalogs of all protein:protein interactions (e.g., yeast two hybrid interactions; protein chips; co--IP; affinity chromatography; resolution of complex mixtures purified from cells) ii) systematic catalogs of biochemical interactions, eg., protein kinase/substrate interactions
c) application of structural biochemistry to genomics: classifying proteins by their shapes
Why proteomics?
1) Expression of many proteins does not correlatewith mRNA levels (in yeast estimated that for50-60% of proteins there is not a linear correlationbetween protein expression and mRNA expression)
2) Many proteins are expressed in an inactive formand only activated post-translationally
3) Many proteins function as part of a complex, andmRNA expression will not reveal these interactions
Mass Spectrometry is the Key Technology for Proteomics
Separates Ions in the Gas Phase Based on mass/charge (m/z) ratio
See this web site for detailed information about Mass Spec:
http://info.med.yale.edu/wmkeck/
1. Determining the Proteome of Cells
In gel
Peptide “fingerprint”
OR
2D Gel Electrophoresis is Coupled with One of Two Types of MS
Peptide Mixture
MALDI (Matrix Assisted Laser Desorption Ionization) Mass Spec
The Actual Mass Spectrum is Compared to Theoretical Mass Spectrum Predicted for All Proteins in The Genome
Peptide Separation
Alternative: HPLC Separation of Peptides
Compare peptide mass + “sequence tags” to all possible patterns in the database
Nanospray Tandem Mass Spec (MS/MS)
MS/MS Techniques Can also be Used to Detect Protein Modification(e.g., phosphorylation. acetylation, etc.)
PI
MW
The 2D Gel is limiting: Rare Proteins or Proteins with ExtremePI (or MW) may not be detected
Comparing the Proteomes of cells Under Different Conditions
2. Cataloging Protein:Protein Interactions
a. Mass Spec to Determine Protein:Protein Interactions
Wave of the Future:Determining the Identity of all Proteins inComplicated Mixtures
B. High Throughput Assays to Determine Protein:Protein Interaction I:Yeast Two-Hybrid Assay
B. High Throughput Assays to Determine Protein:Protein Interaction II:
Protein Chips
Comparison of Different Media for Protein Chips
Protein Chips can also be Used to Study Biochemical reactions:e.g., to Idenitfy Protein Kinase Substrates.
C. Fluorescent Resonance Energy Transfer (FRET) to Study Protein:Protein Interactions Inside Cells
GFPFluorescence
High Throughput Assays can be Developed FRET
3. Application of Structural Biology to Genomics:Predicting Protein Function Based on Protein Shape
Conserved Primary Sequences in Protein Family=
Conserved Secondary Structure=
CONSERVED TERTIARY PROTEIN FOLDING
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