Functional Genomics-Proteomics (I): Overview & Strategies for protein separation. Yao-Te Huang Aug 6, 2010. The website for downloading lecture notes/lecture materials. http://mail.cmu.edu.tw/~ythuang/teaching.summer.2010.htm. Proteome.
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Functional Genomics-Proteomics (I): Overview & Strategies for protein separation
Aug 6, 2010
Part One: Two-Dimensional Gel Electrophoresis (2DGE)
The first dimension:
The second dimension:
The conventional way of doing a typical proteomic experiment.
Part Two: Multi-Dimensional Liquid Chromatography
Discontinuous MDLC or off-line coupling
Case a: Integrated hybrid
Case b: direct serial connection
of two separate columns
Case c: valved connection
with one two-way six-port
valve allowing bypass of first
Possible configurations for 2D-HPLC (contd.)
Case d: valved connection with two two-way six-port valves allowing isolation of first dimension and selected isolation of fraction on trapping column (T).
Case e: valved connection with one two-way six-port valve for selecting or isolating first dimension and two multi-way multi-port valves for selecting or isolating trapping columns 1-8.
Peptide mixtures loaded onto the
ion exchange resin are eluted
using a stepped gradient of salt
,resulting in the release of
first-dimension fractions into the
reverse-phase resin. Second
-dimension fractions are then
eluted from the reversed-phase
resin into the mass spectrometer
using a gradient of acetonitrile.
When this method was applied to the
yeast proteome, over 5000 peptides could be
assigned to a total of 1484 yeast proteins,
representing about (1/4) of the yeast proteome.