Transcript proteomics notes

11-04-11 Proteomics
The science of proteomics
Applications of proteomics
Proteomic methods
a. protein purification
b. protein sequencing
c. mass spectrometry
The proteome is the entire set of proteins expressed
by a genome, cell, tissue or organism
More specifically, it is the set of expressed proteins
in a given type of cells or an organism at a
given time under defined conditions
1. The cellular proteome - the collection of proteins in
a particular cell type under a particular set of
conditions, eg. exposure to hormone stimulation
2. The complete proteome - complete set of proteins
from all of the various cellular proteomes
3. Subsets: the mitochondrial proteome, the viral
proteome, etc.
The bacterial proteome is roughly equivalent to the
bacterial genome; eukaryotic proteomes are much
larger than eukaryotic genomes
1. Alternative splicing
2. Protein modification
In addition to the sequence information in proteins
the proteome can also include
1. Protein folding
2. Functional interactions between proteins and other
molecules
Tools for proteomic research
1. Polyacrylamide gel electrophoresis, to separate proteins
a. SDS gel electrophoresis separates proteins by mass
b. Isoelectric focusing separates proteins by charge
c. Two-dimensional gel electrophoresis (2D PAGE)
d. Special methods, eg gel electrophoresis in the
presence of urea, low pH and Triton X-100
2. Cell fractionation
3. Various methods for protein purification
4. Protein sequencing
5. Mass spectrometry of peptides and intact proteins
6. Yeast two-cell hybrid system - protein-protein interactions
Applications of proteomics
1. Comparison of protein sequences to establish similarities
that define protein families
2. Comparison of the same protein in different species to
reveal evolutionary relationships
3. Search and discovery of common motifs in proteins that
define functions, destinations or processing
4. Sequence data allow a molecular understanding of
diseases
5. Maps of protein interactions help define critical steps in
cellular metabolism
6. Proteomic data can inform treatment of disease
Cell Fractionation
Differential centrifugation permits separation of
different cellular components following cell
disruption
Cell disruption yields a homogenate
Centrifugation of the homogenate yields fractions
called the pellet and supernatant
Differential centrifugation: The supernatant may
again be centrifuged at higher speed to yield
another pellet.
The fraction used is called the crude extract
Subsequent purification of a specific protein
fraction requires a specific assay
1. Enzyme assays are usually quick and easy
NADH absorbs
light at 340 nm
2. The degree of purification is followed by the
specific activity (specific protein/total protein)
Salting in (or out) exploits the solubility of a
protein in different salt concentrations
Dialysis allows removal (or addition) of small molecules
that are permeable to the dialysis membrane
Exclusion chromatography separates proteins
according to their molecular weights
Ion exchange chromatography separates proteins
according to their molecular charge
Affinity chromatography separates proteins
according to their affinity for specific ligands
High Pressure Liquid Chromatography (HPLC)
A high pressure version of liquid chromatography
providing greater speed and resolution
The elution of proteins from
chromatography columns
is often monitored by
UV light absorbance
SDS gel electrophoresis depends on the ability of the anion
detergent sodium dodecyl sulfate to dissolve proteins
SDS coats the protein, causing it to be denatured to a
uniform ellipsoidal shape that is coated with a uniform
negative charge called a micelle
Proteins dissolved in SDS exist as monomers that can then
be separated by electrophoresis through a polyacrylamide
matrix that serves to sieve the proteins according to their
molecular weights
Small proteins migrate more rapidly
Large proteins migrate more slowly
Following electrophoresis
the gel is removed from
the apparatus and stained
to reveal the proteins
If the protein samples are
run in parallel with proteins
of known molecular weight
the molecular size of the
unknown proteins can be
estimated
Isoelectric focusing separates proteins
according to their isoelectric point
Two-dimensional polyacrylamide electrophoresis
(2D PAGE) provides very high resolution of complex
protein mixtures
SDS gel electrophoesis is often used to
follow the progress of a protein purification
The amino acid composition of a protein
can be determined by complete acid hydrolysis
For example: Ala-Gly-Asp-Phe-Arg-Gly
The protein is hyrdolyzed to free amino acids,
which are then reacted with fluorescamine
Edman Degradation is used to determine the
primary structure of a protein
The number of amino acids that can be determined by
Edman Degradation is limited to about 50 amino acids.
Larger proteins are enzymatically or chemically broken into
fragments and sequenced individually
Protein-protein interactions can be detected using
the yeast two-hybrid system
http://en.wikipedia.org/wiki/Two-hybrid_screening