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Lecture 9. Functional Genomics at 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)DNA
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Lecture 9. Functional Genomics at 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)DNA 2)RNA 3) Protein 4) Whole organism 5) Society Lander, E. 1996. The New Genomics: Global Views of Biology. Science 274: 536-539.
Goals of Proteomics a) monitoring the expression and modification state of all proteins in a cell; comparison of proteomes between 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 correlate with mRNA levels (in yeast estimated that for 50-60% of proteins there is not a linear correlation between protein expression and mRNA expression) 2) Many proteins are expressed in an inactive form and only activated post-translationally 3) Many proteins function as part of a complex, and mRNA expression will not reveal these interactions
1. Determining the Proteome of Cells 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/
2D Gel Electrophoresis is Coupled with One of Two Types of MS Peptide “fingerprint” In gel OR
MALDI (Matrix Assisted Laser Desorption Ionization) Mass Spec Peptide Mixture
The Actual Mass Spectrum is Compared to Theoretical Mass Spectrum Predicted for All Proteins in The Genome
Nanospray Tandem Mass Spec (MS/MS) Compare peptide mass + “sequence tags” to all possible patterns in the database Peptide Separation Alternative: HPLC Separation of Peptides 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 Extreme PI (or MW) may not be detected
2. Cataloging Protein:Protein Interactions a. Mass Spec to Determine Protein:Protein Interactions Wave of the Future: Determining the Identity of all Proteins in Complicated 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
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 GFP Fluorescence 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=