Proteomic analysis of colorectal cancer: prefractionation strategies using two‐dimensional free‐flow electrophoresis

Abstract

This review deals with the application of a new prefractionation tool, free-flow electrophoresis (FFE), for proteomic analysis of colorectal cancer (CRC). CRC is a leading cause of cancer death in the Western world. Early detection is the single most important factor influencing outcome of CRC patients. If identified while the disease is still localized, CRC is treatable. To improve outcomes for CRC patients there is a pressing need to identify biomarkers for early detection (diagnostic markers), prognosis (prognostic indicators), tumour responses (predictive markers) and disease recurrence (monitoring markers). Despite recent advances in the use of genomic analysis for risk assessment, in the area of biomarker identification genomic methods alone have yet to produce reliable candidate markers for CRC. For this reason, attention is being directed towards proteomics as a complementary analytical tool for biomarker identification. Here we describe a proteomics separation tool, which uses a combination of continuous FFE, a liquid-based isoelectric focusing technique, in the first dimension, followed by rapid reversed-phase HPLC (1–6 min/analysis) in the second dimension. We have optimized imaging software to present the FFE/RP-HPLC data in a virtual 2D gel-like format. The advantage of this liquid based fractionation system over traditional gel-based fractionation systems is the ability to fractionate large quantity protein samples. Unlike 2D gels, the method is applicable to both high-Mr proteins and small peptides, which are difficult to separate, and in the case of peptides, are not retained in standard 2D gels.

Modeling the Isoelectric Focusing of Peptides in an OFFGEL Multicompartment Cell

“…3 But because further protein analysis and characterization by mass spectrometry 4 require tedious sample preparation, new IEF schemes and devices have been designed for prefractionation of proteins by IEF: 5,6 several teams have explored the use of free-flow electrophoresis for the fractionation of proteomic samples. [7][8][9][10][11][12] Righetti et al have introduced multicompartment electrolyzers, in which proteins are separated into different compartments separated by Immobiline membranes. [13][14][15] Wall et al have also validated the use of Rotofor for fractionation of proteins prior to RP-HPLC and MALDI-TOF analysis of intact proteins.…”

Recent developments in CE and CEC of peptides

“…Free-flow electrophoresis, particularly the FF-IEF mode has been used for prefractionation of complex peptide/protein mixtures in proteomic studies of cells [235][236][237] and body fluids, such as human plasma [238] and human saliva [239].…”

Proteomic analysis reveals overexpression of moesin and cytokeratin 17 proteins in colorectal carcinoma

“…The angiogenesis inhibitor bevacizumab, a humanized VEGF blocking antibody, demonstrated a marked inhibition of tumor angiogenesis and tumor growth and is now widely used in clinical practice for the treatment (8)(9)(10). Other approaches for targeted therapy of colorectal cancer include the inhibitors of Src tyrosine kinases (11)(12)(13)(14), mTOR (15) or PI3K (16,17), and molecules inducing apoptosis (18)(19)(20)(21)(22), and radioimmunotherapy using cancer-specific antibodies (23). However, the benefit from cetuximab and/or bevacizumab treatment is so limited that only a small proportion of patients respond to the drugs and their lifespan is increased by only a few months compared to those who only receive supportive care.…”