Proteome and Gene Expression Analysis

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Proteome and Gene Expression Analysis

• Chapter 15 16

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The Goals

• Functional Genomics
• To know when, where and how much genes are
  expressed.
• To know when, where, what kind and how much of
  each protein is present.
• Systems Biology
• To understand the transcriptional and
  translational regulation of RNA and proteins in
  the cell.

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Genes and Proteins

• First, well talk about how to find out what
  genes are being transcribed in the cell.
• This is often referred (somewhat misleadingly) to
  gene expression.
• Second, well look at measuring the levels of
  proteins in the cell.
• The real expression of protein coding genes
• Third, well talk about how we process and
  analyze the raw data using bioinformatics.

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Review Gene Arrays

• Put a bunch of different, short single-stranded
  DNA sequences at predefined positions on a
  substrate.
• Let the unknown mixture of tagged DNA or RNA
  molecules hybridize to the DNAs.
• Measure the amount of hybridized material.

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Getting the Data
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Getting Protein Expression Data

• To be able to understand protein expression, we
  need the concentrations of all proteins (the
  proteome) in difference cell and tissue types
  under varying conditions.
• Large scale identification of proteins is much
  more limited than for RNA.
• Nothing really equivalent to RNA expression
  microarrays or high-throughput sequencing exists
  yet.
• Relatively low-throughput technologies are all
  that we have right now.

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Measuring Protein Expression

• In order to measure all the types of protein in a
  cell we must
• Extract the proteins
• Purify the proteins
• Identify the individual proteins
• How do we accomplish purification and
  identification of proteins.

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The TechnologiesProtein Expression

• Low-throughput
• 2D Gel Electrophoresis Mass Spectrometry
• Liquid chromatograph Mass Spectrometry
• Protein microarrays
• Limited in application at this point
• Can be used for things other than protein
  expression like protein-protein interactions

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Extracting the Proteins

• First, the proteins are extracted from the cells
  using lysis.
• This involves a detergent that destroys the
  membranes of the cell.

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Separating the Proteins2D Gel Electrophoresis

• First step pI/pH
• Proteins are introduced to a gel with an
  imobilized pH gradient.
• A charge is applied.
• Proteins migrate until the pH causes them to lose
  their charge (isoelectric point) and then stop.
• Second step mass
• First gel transferred to second gel
• SDS (detergent) breaks structure and charges the
  proteins proportional to their mass.

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Using the 2D Gel

• Staining makes the spots containing the
  individual (we hope) proteins visible.
• The gel is photographed.
• Protein level (concentration) can be estimated by
  image processing.
• Individual, stained spots can be cut out for
  evaluation by Mass Spectrometry.

segmenting
dust
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Two channel 2D Gels

• Low signal-to-noise is a problem with protein
  gels, as it is with RNA expression arrays.
• A similar trick of putting two cell lysates
  (samples) on one gel can help.
• Registration problems and sample-dependent
  effects are thereby minimized.
• However, 1-channel gels allow comparing more than
  two samples

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Protein Identification Using Mass Spectrometry
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Steps of Mass Spectrometry

• Digest
• Sample (spot) is digested with a proteolytic
  enzyme
• Spectrum
• Peaks correspond to the mass-charge ratio of
  protein fragments
• These provide a fingerprint
• Identify
• Compare fingerprint to theoretical fingerprints
• Post-translational modifications screw things up.

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Spectrum Protein Fingerprint
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Tricks Protein chips

• If you had an antibody to every possible protein
  and could put it on a chip, and you could label
  the proteins in your sample, you would have
  something equivalent to an RNA expression
  microarray.
• Getting reliable antibodies is difficult and
  expensive.
• Arrays with 500 to 2000 proteins are available
  commercially Clontech, Eurogentec, Arrayit etc.

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Not part of this subject, but cool
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Protein Arrays for Measuring Protein-Protein
Interactions

• You can synthesize proteins from DNA directly on
  a substrate.
• Nano-well approach
• Printing approach DAPA (DNA to Protein Array)
• These can be used for measuring binding between
  proteins, but not for identification of proteins.

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Next timeAnalyzing Gene and Protein Expression
Data
Protein Expression Clustering
Gene expression clustering