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

Abstract

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.

Proteome Analysis of Microdissected Formalin-fixed and Paraffin-embedded Tissue Specimens

“…For profiling tryptic peptides obtained from an FFPE tissue sample, the entire content of focused peptides in the CIEF capillary was split into 19 individual fractions (Figure 2), which were further resolved by nano-RPLC and identified using nano-ESI-LTQ-MS/MS. The number of distinct peptide identifications measured from each CIEF fraction is significantly greater than that typically reported in the literature using other IEF techniques, including immobilized pH gradient gels (Cargile et al 2004;Essader et al 2005) and gel-free approaches (Wall et al 2000;Zuo et al 2001;Yan et al 2003;Moritz et al 2004) such as chromatofocusing, immobilized pH membranes, Rotofor, and free-flow electrophoresis. A key feature of our CIEF-based multidimensional separation technology is the elimination of protein/peptide loss and dilution in an integrated platform while achieving comprehensive and ultrasensitive analysis of protein expression profiles within FFPE tissue specimens.…”

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

“…The quantitative proteomic profiling of proteins using multidimensional chromatography has also been performed (reviewed in [20]). A number of proteomic studies have been performed utilising such techniques [21][22][23]. More recently, multidimensional protein separations have been performed integrating both 'top down' and 'bottom up' approaches for protein identifications [24].…”

Capillary separations enabling tissue proteomics‐based biomarker discovery

“…Besides the ultrahigh resolving power of CIEF-based multidimensional separations, initial proteome studies [46,47] have supported the ability to perform comprehensive analysis of yeast cell lysates while requiring the amount of yeast peptides or proteins that are two to three orders of magnitude less than those utilized in a multidimensional LC system [33][34][35][36][37][49][50][51]. Such an increase in proteome sensitivity resulting from IEF concentration and separation of peptides and proteins is further supported by the study of Cargile et al [52] using IPG gels.…”