A Comparison of Drug-Treated and Untreated HCT-116 Human Colon Adenocarcinoma Cells Using a 2-D Liquid Separation Mapping Method Based upon Chromatofocusing PI Fractionation
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- 作者：Fang Yan ; Balanehru Subramanian ; Alexander Nakeff ; Timothy J. Barder ; Steven J. Parus ; and David M. Lubman
- 刊名：Analytical Chemistry
- 出版年：2003
- 出版时间：May 15, 2003
- 年：2003
- 卷：75
- 期：10
- 页码：2299 - 2308
- 全文大小：249K
- 年卷期：v.75,no.10(May 15, 2003)
- ISSN：1520-6882
文摘
A multidimensional chromatographic 2-D liquid-phaseseparation method has been developed for differentialdisplay of proteins from cell lysates and applied to acomparison 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 columnsin the first dimension followed by separation of theproteins in each pI fraction using nonporous reversed-phase HPLC. A 2-D map of the protein content of eachcell line based upon pI versus hydrophobicity as detectedby UV absorption was generated and a differential displaymap indicating the presence of up- or downregulatedproteins displayed using ProteoVue and DeltaVue software. Using this method, >1000 protein bands could bedetected in 0.2 pH fractions over a pH range of 4-7. Inaddition, the liquid eluent from the separation wasdirected on-line into an electrospray TOF-MS to obtain anaccurate molecular weight of the intact proteins. Anaccurate molecular weight together with the peptide mapwas used to obtain protein identification using databasesearching. The method has been shown to have highreproducibility for quantitative differential display analysisof interlysate comparisons, generation of accurate proteinidentifications, and ease of data interpretation. It has beenused herein to identify proteins that change as a functionof drug treatment. The relative simplicity of the currentprocedure and the potential for full automation will makethis technique an essential tool in future proteomicstudies.