Molecular Biologists Guide to Proteomics

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Molecular Biologists Guide to Proteomics

• Christopher Kvaal
• Montana State University-Bozeman

Source Molecular Biologist Guide to Proteomics,
Graves, P.R. and Haystead, T.A.J., Micro Mol Biol
Rev. Mar 2002 pp 39-63
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Topics of Discussion

• Review
• Introduction to Proteomics
• Technology of Proteomics
• Applications of Proteomics

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Review

• Bioinformatics
• Storing, Organizing, and Analyzing Sequence Data
• Functional Genomics
• Contextual Use of Bioinformatic Data
• i.e. DNA Array or SAGE
• Proteomics

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Why do we have these new disciplines?

• To much information
• What do we do with all the sequence data?
• 45 microorganism genomes have been sequenced and
  170 more are in progress
• 5 eukaryotes have been completed
• Saccharomyces cerevisiae
• Schizosaccharomyces pombe
• Arabodopsis thaliana
• Caenorhabditis elegans
• Drosophilia melanogaster
• Rice, Mouse and Human are nearly done
• 2/3 of all genes identified have no known
  function

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Move over Genomeon to Proteomics

• If the genome is the blueprint of an
  organism---who reads it?
• The proteome is all the proteins that are called
  for by the genome
• At this point no computer algorithm can do this
• A computer can decode all 6 reading frames of an
  organism
• A computer can compare these.. But then what?

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Genomic DNA

• Structure
• Regulation
• Information
• Computers cannot determine which of these 3 roles
  DNA play solely based on sequence (although we
  would all like to believe they can)

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Introduction to Proteomics

• Definitions
• 1. Classical - restricted to large scale
  analysis of gene products involving only proteins
• 2. Inclusive - combination of protein studies
  with analyses that have genetic components such
  as mRNA, genomics, and yeast two-hybrid
• Dont forget that the proteome is dynamic,
  changing to reflect the environment that the cell
  is in

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1 gene 1protein?

• 1 gene is no longer equal to one protein
• In fact, the definition of a gene is
  debatable..(ORF, promoter, pseudogene, gene
  product, etc)
• 1 genehow many proteins

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Why Proteomics?
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Why Proteomics?

• Annotation of genomes, i.e. functional annotation
• Genome proteome annotation
• Protein Function
• Protein Post-Translational Modification
• Protein Localization and Compartmentalization
• Protein-Protein Interactions
• Protein Expression Studies
• Differential gene expression is not the answer

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Cell Specific Expression

• All cells in a metazoan organism contain the same
  genomic DNA
• How does a genome project answer the question of
  why liver specific genes are not expressed in
  brain?

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Types of Proteomics

• Protein Expression
• Quantitative study of protein expression between
  samples that differ by some variable
• Structural Proteomics
• Goal is to map out the 3-D structure of proteins
  and protein complexes
• Functional Proteomics

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Technology of Proteomics

• Separation and Isolation of Proteins
• 1D and 2D PAGE
• Edman Sequencing
• Mass Spectrometry
• Database utilization

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Technology of Proteomics

• How do you separate proteins.PAGE
• Has not changed in the 32 years since its
  inception..the question answered is still the same

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Technology of Proteomics

• Edman (N-terminal) Sequencing
• Introduced in 1949
• Run PAGE then Sequence
• Alternative to PAGE
• Digest whole protein mixture
• Start sequencing
• Only works with High Abundance Proteins

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Technology of Proteomics

• Mass Spectrometry (MS)
• Enables peptide mass and sequence to be obtained
• Faster, more sensitive, more difficult and more
  expensive

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Applications of Proteomics

• Characterization of Protein Complexes
• Protein Expression Profiling
• Yeast Genomics and Proteomics
• Proteome Mining
• Protein Arrays

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Proteome Mining

• A functional proteomics approach
• A Proteome Mine Example
• ATP is immobilized to beads in protein kinase
  conformation
• Total protein is mixed the beads and the mixture
  washed
• Remaining proteins isolated and
  identifiedprotein kinases, and purine dependent
  metabolic enzymes
• Immobilize a putative drug to bead and test for a
  cellular ligand

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Yeast Genomics and Proteomics

• First example of the 2-omics used in a
  complementary fashion
• Comparative proteome experiment identified
  over-expressed proteins
• Protein info used to design genetic experiments
  and verify finding

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Protein-Protein Interactions

• Yeast two-hybrid system
• Establish the role of a protein with a guilt by
  association argument
• Organism wide (S. Cerevisiae) comparision
• 6000 ORFs systematically compared in a matrix
• Are we crazy, how to interpet the results

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Protein Arrays

• Another Functional Proteomics Approach
• Same concept as a DNA Array
• Has been published in a peer-reviewed journal

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Challenges of Proteomics

• The Native-conformation question
• Proteins cannot be studied with the scale, speed,
  sensitivity and reliability that nucleic acids
  achieve
• Low-abundance proteins may be the most important
  class of proteins and the most difficult to study
  with current methodologies
• One of you needs to go out and invent Protein
  PCR

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The End