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.
A multidimensional chromatographic 2-D 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 HPLC. A 2-D 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 downregulated proteins displayed using ProteoVue and DeltaVue 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 TOF-MS to obtain an accurate molecular weight of the intact proteins. An accurate molecular weight together with the 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, 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. The relative simplicity of the current procedure and the potential for full automation will make this technique an essential tool in future proteomic studies.
Yan
Fang
F
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Subramanian
Balanehru
B
Nakeff
Alexander
A
Barder
Timothy J
TJ
Parus
Steven J
SJ
Lubman
David M
DM
D000230:Adenocarcinoma / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D009363:Neoplasm Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D009363:Neoplasm Proteins