Proteonomics

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Proteonomics

• A Style of Biological Analysis in which the
  protein content of an entire genome or in an
  entire organisim is studied
• An extention of the human genome project
  Human Proteome Organization HUPO

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• HUPO Goals -Learn the following about every
  protein!
• Biological Process
• Molecular Function
• Cellular Component

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• The Tools
• Protein Arrays-- look for protein/ protein
  interaction
• 2-D Gels
• Yeast Two Hybridizatoin
• Protein Sequencing and Maldi-Tof

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2- D Electrophoresis
1. Isoelectric Focusing 2. SDS-PAGE
1 - Outer Buffer Chamber 2 -
complete Slab Gel System 3 - Central
Running Module 4 - Electroblotter
Module 5 - 2-D Capillary Module
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Amphoteric Molecules Form the ph Gradient
As Protein Travels through to Cathode Charge on
Protein Changes
COOH NH3
COO- NH2
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Ventrial
Atrium
http//userpage.chemie.fu-berlin.de/pleiss/dhzb.h
tml
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Edman Sequencing Cost 150
The Edman chemistry cycle consists of four
stages 1. Coupling The N-terminus of the
protein couples with PITC to form a
phenylthiocarbamyl (PTC)-polypeptide.
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2. Cleavage - Group on AA becomes unstable
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3. Conversion to a more stable group
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Problem N-terminus can be blocked
http//www.biotech.iastate.edu/facilities/protein/
nsequence494.html
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Method for FingerPrinting Proteins
1. Break Protein into Peptides with the chemical
Trypsin. 2. Anneal Peptides to Matrix and Ionize
with a Laser.
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3. Peptides Travel Down Flight Tube and Time of
Flight is Recorded. Each protein has a unique
fingerprint that can be Compared to the database
to determine what the protein is.
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(matrix-assistedlaser/desorption ionisation, time
of flight analyser)
http//www.biotech.iastate.edu/facilities/protein/
maldi.html
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• Two Hybrid Systems
• System to look at Protein Interactions
• Uses the principles of protein activation that we
  have
• Learned already

• Clone your protein of interest
• On a DNA binding domain

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B. Place in Cell with a Reporter gene that has a
Binding site in its DNA for this binding
element. - Nothing should happen because no
activation domain
Viral promoter Reporter Gene
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C. Create Library of all other genes in the
genome cloned Next to an activation domain D.
Transform cells with only One of these genes
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Assay for Reporter Gene Activation. Find out
what protein bound yours to get clues about what
it does.
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Problems - sometimes Requires Modification To
Interact