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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Aug 6, 2010
The first dimension:
The second dimension:
Part Two: to sample complexityMulti-Dimensional Liquid Chromatography
Discontinuous MDLC or off-line coupling to sample complexity
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.) to sample complexity
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 completely retained on the first dimension. It is then eluted, in discrete fractions, directly onto the second dimension. While the initial eluate is being separated in the second dimension, the rest of the sample remains in the first dimension until it, in turn, is eluted in a stepwise manner and separated in the second dimension.
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.