Transcript Proteomics – 2D gels - Department of Chemistry and Biochemistry

Proteomics:
Its Function and Methods
Ryan Victor
Proteomics
What Is It?
What’s Being Studied?
How Is It Done?
Two-Dimensional Electrophoresis
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.
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 it’s 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.
What’s 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.
How to Study It
Antibody Method:
1) Antibodies are adding to the protein mixture
2) Antibodies bind to proteins that have modified
3) Proteins of interest can be separated based on the
modification.
2 Dimensional Protein Gels
-What’s the Difference?
-How’s It Done?
-What Can We Do With It
Fluorescently labeled proteins in difference gel electrophoresis
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.
How It’s Done
We’ll 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.
How It’s 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.
How It’s 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.
How It’s 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.
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
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.
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.
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.
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-modifications.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