Proteome Analysis

July 1, 2009

Learning objectives. Understand the theoretical basis of 2D-SDS Gel Electrophoresis. Understand the latest technique for identifying proteins using mass spectrometry. Become familiar with the SWISS-2DPAGE Database

Workshop on identifying proteins with the pI, MW and a few peptide masses.

What is the proteome?

genome

transcriptome

proteome

metabolome

2D-SDS Gel ElectrophoresisWhat is it?

This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another.

Proteins separate over a large area, having a greater chance of resolution.

First dimension

Separation by isoelectric point

Isoelectric point-pH at which the net charge on the molecule is zero

Visuals

http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%203

Figure 4-44. Alberts et al. Molecular Biology of the Cell, 3rd edn.

2nd Dimension-to separate by size

Five steps:1. Isolate proteins

2. Denature proteins

3. Load onto gel

4. Apply voltage gradient

5. Stain

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

Figure 4-44. Alberts et al. Molecular Biology of the Cell, 3rd edn.

Figure 3-42. Lodish et al. Molecular Cell Biology.

Databases of 2D-Gels

http://www.umh.ac.be/~biochim/balhf.htm

http://www-lmmb.ncifcrf.gov/EP/table2Ddatabases.html

http://www.weihenstephan.de/blm/deg/manual/manualwork2html02testp9.htm

Laser

Detector

Detector

m/z

Ab

%975.43

Vacuum

Plate

Linear Mode

Reflection Mode

http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS

Protein Prospector

Another Web-based program is Protein Prospector (UCSF)

http://prospector.ucsf.edu/