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| Institute: |
Lab or Branch |
| Eprogen, Darien, IL |
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| Title: |
| A Comparison of Drug-Treated and Untreated
HCT-116 Human Colon Adenocarcinoma Cells Using a 2D Liquid
Separation Method |
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| Authors: |
| F. Yan, B. Subramanian, A. Nakeff, T. J.
Barder, S. J. Parus, D. M. Lubman |
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| Abstract: |
| A multidimensional chromatographic 2D liquid-phase
separation method has been developed for differential
display of proteins from cell lysates and applied to a
comparison of protein expression between Peninsularinone-treated
and untreated HCT-116 human colon adenocarcinoma cells.
The method involves fractionation according to pI using
chromatofocusing with analytical columns in the first
dimension followed by separation of the proteins in each
pI fraction using nonporous reversed-phase (NPS-RP) HPLC.
A 2D map of the protein content of each cell line based
upon pI versus hydrophobicity as detected by UV absorption
was generated and a differential display map indicating
the presence of up- or down-regulated proteins displayed
using ProteoVue(tm) and DeltaVue(tm) software. Using this
method, >1000 protein bands could be detected in 0.2
pH fractions over a pH range of 4-7. In addition, the
liquid eluent from the separation was directed on-line
into an electrospray (ESI) TOF-MS to obtain an accurate
MW of the intact protein. The pI range together with an
accurate MW and a peptide map was used to obtain protein
identification using database searching. The method has
been shown to have high reproducibility for quantitative
differential display analysis of interlysate comparisons,
the generation of accurate protein identifications and
ease of data interpretation. It has been used herein to
identify proteins that change as a function of drug treatment.
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