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Proteomics

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Proteomics: Its Function and Methods Ryan Victor Proteomics What is It? What is It? What s Being Studied How to Study It 2 Dimensional Protein Gels The Difference ... – PowerPoint PPT presentation

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Title: Proteomics


1
Proteomics
  • Its Function and Methods

Ryan Victor
2
Proteomics
What Is It? Whats Being
Studied? How Is It Done? Two-Dimensional
Electrophoresis
3
What is It?
Proteomics is the study of proteins that are
generated from the genetic code of an organism.
Proteomics differs from genomics in that the
chromosomes for a genome are consistent
throughout a multicellular organism, protein
output varies from cell to cell based on the
cells function.
While the genetic code for humans has been
completed through the human genome project,
proteomics will be a much longer, more intensive
and time consuming study.
4
What is It?
Initial attempts to study proteins used mRNA as
the determining factor for protein level output.
However, post translational modifications make
the method invalid.
The study of proteomics is forced to focus on
very stringent conditions in a cell in order for
the results to be valid.
The cell must change its protein output based on
its level of stress and metabolic needs. The
results of a proteomics study can really only be
generalized to the conditions that the cell faced
during the study.
5
Whats Being Studied
Phosphorylation often is used to activate a
protein, usually on a serine or threonine
residue. Activation occurs as the
phosphorylation is used to signal other proteins
and thus permit binding between them.
Ubiquination often is a signal for a protein to
be degraded. Once a protein has ubiquitin
attached to it, it can be labeled for destruction
by proteasome.
6
How to Study It
Antibody Method
  • Antibodies are adding to the protein mixture
  • Antibodies bind to proteins that have modified
  • Proteins of interest can be separated based on
    the modification.

7
2 Dimensional Protein Gels
-Whats the Difference? -Hows It Done? -What
Can We Do With It
Fluorescently labeled proteins in difference gel
electrophoresis
8
The Difference
Conventional protein gels only run in one
dimension, as the name implies, 2D gels run in 2
dimensions.
2nd running dimension allows separation based on
a 2nd characteristic.
Common separation characteristics are isoelectric
point in the first dimension, followed by
mass/size separation in the 2nd dimension.
9
How Its Done
Well look at isoelectric focusing as an example
Proteins are fed into a gel medium that has a pH
gradient to it. pH gradient is formed by adding
polyampholytes to the gel medium or using a
gradient gel. Polymapholytes are much like amino
acids in that they are zwitterionic.
10
How Its Done
Proteins each have their own isoelectic point
that comes about as an average of their amino
acid isoelectic point. Proteins are charged at
different pHs. When they reach the region of
the gel that matches their isoelectric point,
they stop moving. Proteins can be removed at
this point for further analysis if
desired. Proteins can also be subjected to
further analysis within the gel.
11
How Its Done
Now that the proteins have been separated by
isoelectic point, they can be analyzed based on
their mass.
Proteins are separated by mass using Sodium
Dodecyl Sulfate. SDS acts as a detergent to
uncoil the protein and give it a negative charge,
since the proteins have zero charge after the
isoelectic focusing.
The proteins migrate by applying an electric
field 90 degrees from where it was located for
isoelectric focusing.
Proteins have now migrated in 2 different
dimensions from their starting point, giving a
3D gel.
12
How Its Done
Now that the proteins have migrated to their new
positions based on isoelectric point and mass,
they can be analyzed.
Gel can be stained using coomassie or silver.
Silver binds to the cysteine groups in the
protein, and leaves a dark stain on the gel after
development.
Coomassie binds to arginine, histidine, and
aromatic amino acids. It can also be used to
replace SDS to give the negative charge to the
protein.
13
Application
Comparing the protein output between two
different organism samples Comparing output
between cells within a specific
organism Determining whether an organism lacks a
specific protein output Associating protein
output with a specific disease, and thus aiding
in drug development
14
Problems
Two dimensional gels must still be read, which
has its own variety of difficulties
- Spots can overlap - Gel may not be
reproducible - Spots may not properly visualize
and be too weak to see.
Duplicate gels overlaid upon each other in the
Delta 2D program
Scanners and computer programs are used to read
the gels. They are only as good as the scanners
acuity and the programs ability to discern spots
on the scan.
15
Difference Gel Electrophoresis
Fluorescent dye is added to the proteins, then
the gel is analyzed using a laser to excite the
fluorescent dyes.
The abundance of proteins can be viewed from
different samples to give an idea of the
difference between them.
Eliminates the difficulty of comparing different
gels.
16
Summary
Proteomics allows researchers to study the
actual output of the cells rather than just the
DNA blueprints. Proteomics can be used to
develop effective treatments for
diseases. 2-Dimensional Electrophoresis is an
effective method for visualizing the results of a
proteomics experiment. 2D gels are not without
their difficulties.
17
References
Unlu, M., Morgan, M., Minden J. (1997).
Difference Gel Electrophoresis. Electrophoresis
11, 2071-7
King, M. (2009, November 2nd). Protein
Modifications. Retrieved March 5th 2009 from The
Medical Biochemistry Page website,
http//themedicalbiochemistrypage.org/protein-modi
fications.html Anderson, N. (2005). Proteome and
Proteomics. Electrophoresis 19, 1853-1861 Maiman
Institute for Proteome Research. Two-Dimensional
Gel Electrophoresis. Retrieved March 5th 2009
from the Maimane Institute for Proteome Research
at Tel Aviv website, http//www.tau.ac.il/lifesci/
units/proteomics/2dimgel.html Berth, M. et al.
(2007) The State of the Art in Analysis of
Two-Dimensional Gel Electrophoresis Images.
Applied Microbiology Biotechnology 76, 1223-43
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