Proteomic Target Finding

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• Proteomic Target Finding

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Genomics
protein
polysomal RNA
regulatory step 3
regulatory step 1 (polymorphisms)
genomic locus
Poly(A)RNA
transcript
regulatory step 2
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regulatory step 5 post-translational modifications
Proteomics
protein
regulatory step 4
polysomal RNA
genomic locus
Poly(A)RNA
transcript
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One genome - different proteoms
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One genome - different proteoms
"Not only the genome, but in particular the
proteins present the proteome determine the
appearance and state of a biological
organism" (Taken from Lottspeich, F. Angew.
Chem. 1999, 111, 2630-2647)
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What is the Proteome?

• "A proteome represents the protein pattern of an
  organism ... at a certain moment and under
  defined limiting conditions."

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Why are the genome and the proteome often
uncorrelated?

• No biological state exists in which all possible
  proteins of an organism are expressed (different
  gene expression)
• mRNA and protein amount is uncorrelated due to
• mRNA degradation
• translational control
• protein degradation
• posttranslational modifications (e.g.
  glycosylation, phosphorylation etc.)

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Why is genomics much more popular though than
proteomics?

• DNA/RNA can be treated much easier than proteins!
• proteins differ very much in their
  physicochemical properties (solubility,
  stability, charge etc.)
• proteins are much more complex (large structural
  variety)
• no correlate to Gene Chips
• Gene technology has been developed to a very high
  level (e.g. by the human genome project)
• amplification (e.g. by PCR)
• sequencing
• disruption of gene function (e.g. by antisense
  techniques)

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Steps involved in Proteomics

• Sample preparation
• Protein separation
• Quantitative and sequence analysis of protein
  spots
• Database analysis of protein patterns by means of
  bioinformatics
• Protein chemical characterization and analysis of
  posttranslational modifications

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Protein separation is performed by 2D gel
electrophoresis
Cut out individual spotsand determine their
amino acid sequence

• First dimension IEF (isoelectric focusing)
• protein moves through polymer gel with pH
  gradient strips
• protein stops moving in the strip corresponding
  to its isoelectric point
• Second dimension SDS-PAGE (sodium dodecyl
  sulfate-polyacrylamide gel electrophoresis)
• protein is loaded with negatively charged SDS
• protein moves in electric field according to its
  mass

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Quantitative and Sequence Analysis of Protein
Spots

• Immunoblotting (Western Blot)
• Protein hydrolysis and amino acid composition
  analysis
• Microsequencing by Edman degradation
• MALDI-MS

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Microsequencing of proteins by Edman degradation
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Protein analysis is performed by MALDI-MS
(matrix-assisted laser desorption ionisation
mass spectroscopy)
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Differential Proteome Analysis
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Differential Proteome Analysis

• Differences can be measured between
• normal and diseased patients (target finding)
• patients before and after drug treatment
  ("pharmacoproteomics")
• cells in different environmental or developmental
  states