Transcript A PRIMER OF GENOME SCIENCE - Temple University, Center …

A PRIMER OF GENOME
SCIENCE
Chapter 4 Proteomics and
Functional Genomics
Hao Sun
Index
Introduction
Qualitative methodologies
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2D electrophoresis
Mass spectrometry(MS)
Quantitative methodologies
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X-ray crystallography
Nuclear magnetic resonance(NMR) Spectroscopy
The Nobel Prize in Chemistry 2002
John B. Fenn
Koichi Tanaka
Kurt Wüthrich
USA
Japan
Switzerland
•"for the development of methods for identification and
structure analyses of biological macromolecules"
•"for their development of soft desorption ionisation methods
for mass spectrometric analyses of biological
macromolecules“
•"for his development of nuclear magnetic resonance
spectroscopy for determining the three-dimensional structure
of biological macromolecules in solution“
Copyright© 2003 The Nobel Foundation
Introduction
•What is Proteomics?
Proteomics is the study of the structure and expression of proteins and of
the interactions between proteins.
•Focus on how data on the expression and identity of proteins
in cell is obtained.
•Include several methods that are used to study the structure of
proteins.
•Key features: Predicted Protein domains from sequence
alignment.
2D electrophoresis
Proteins are separated in two sequential steps: first by their
charge and then by their mass.
1. Isoelectric focusing (IEF) can resolve proteins that differ
by only one charge unit. Charged proteins will migrate
through the PH gradient until they reach their isoelectric
point.
2. When an electric field is imposed, the proteins will migrate
from the IEF gel into the SDS slab gel and then separate
according to their mass.
3. For example, as many as 1000 proteins can be resolved
simultaneously.
2D electrophoresis
SDS polyacrylamide-gel
electrophoresis (SDS-PAGE)
“Molecular Biology of the
Cell” 3rd ed. Bruce Alberts
Mass spectrometry(MS)
A powerful technique for measuring the mass of proteins
Step1 Pulses of light from a laser ionize a protein or
peptide mixture.
Step2 accelerating the molecular ions.
Step3 detecting the ions.
Mass spectrometry(MS)
Detecting Time is inversely proportional to their mass and
directly proportional to the charge on the protein.
Other qualitative methods for protein
1. Protein Microarry
2. Protein Interaction Maps
3. Gel Filtration Chromatography
4. Ion-Exchange Chromatography
5. Affinity Chromatography
6. Chromogenic and Light-Emitting Enzyme Reactions
7. Western Blotting
8. Characteristics of Different Radiolabels
References:
“A PRIMER OF GENOME SCIENCE” Greg Gibson
“Molecular Cell Biology” 4th ed Harvey Lodish
X-ray crystallography
1.
A narrow beam of xrays strikes a crystal.
2.
part of it passes straight
through and the rest is
diffracted in various
directions.
3.
The intensity of the
diffracted waves is
recorded on an x-ray
film or with a solidstate electronic detector.
X-ray crystallography
Basic pancreatic trypsin inhibitor (BPTI).
Nuclear magnetic resonance(NMR) Spectroscopy
A is a two-dimensional NMR spectrum derived from the enzyme cellulase.
B structures gives a good indication of the probable three-dimensional
structure.
References: “Molecular Biology of the Cell” 3rd ed. Bruce Alberts
“Molecular Cell Biology” 4th ed Harvey Lodish
http://www.rcsb.org/pdb/
Protein Structure Determination
Clone coding sequence into expression vector
Express and purify protein
Verify identity by sequence and biochemical assay
Obtain sufficient protein in appropriate state for phasing
Grow crystals or establish NMR solution
Take X-ray or NMR measurement
Solve structure computationally
Infer functional features and comparative structure
Thank You