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Proteome Analysis

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Proteome Analysis July 1, 2009 Learning objectives. Understand the theoretical basis of 2D-SDS Gel Electrophoresis. Understand the latest technique for identifying ... – PowerPoint PPT presentation

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Title: Proteome Analysis


1
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.

2
What is the proteome?
metabolome
proteome
transcriptome
genome
3
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.

4
First dimension
  • Separation by isoelectric point
  • Isoelectric point-pH at which the net charge on
    the molecule is zero

5
Visuals
http//www.aber.ac.uk/parasitology/Proteome /Tut_2
D.htmlSection203
6
Figure 4-44. Alberts et al. Molecular Biology of
the Cell, 3rd edn.
7
2nd Dimension-to separate by size
  • Five steps
  • 1. Isolate proteins
  • 2. Denature proteins
  • 3. Load onto gel
  • 4. Apply voltage gradient
  • 5. Stain

8
http//www.davidson.edu/academic/biology /courses/
Molbio/SDSPAGE/SDSPAGE.html
9
http//www.davidson.edu/academic/biology /courses/
Molbio/SDSPAGE/SDSPAGE.html
10
http//www.davidson.edu/academic/biology /courses/
Molbio/SDSPAGE/SDSPAGE.html
11
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12
Figure 4-44. Alberts et al. Molecular Biology of
the Cell, 3rd edn.
13
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14
Figure 3-42. Lodish et al. Molecular Cell
Biology.
15
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16
Databases of 2D-Gels
http//www-lmmb.ncifcrf.gov/EP/table2Ddatabases.ht
ml
http//www.umh.ac.be/biochim/balhf.htm
17
http//www.weihenstephan.de/blm/deg/ manual/manual
work2html02testp9.htm
18
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20
Detector
Reflection Mode

Laser
Plate

Linear Mode
975.43
Detector
Ab
Vacuum
m/z
21
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24
http//www.matrixscience.com/cgi/search_form.pl?FO
RMVER2SEARCHMIS
25
Protein Prospector
  • Another Web-based program is Protein Prospector
    (UCSF)
  • http//prospector.ucsf.edu/
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