A colorectal cancer study aimed at identifying potential biomarkers using two parallel workflows has been performed. One workflow was gel-based and utilized 2D electrophoresis and the Ettan DIGE and MALDI-ToF for relative quantification and subsequent protein identification. The other approach included Ion exchange protein prefractionation and subsequent separation of digested protein fractions on a multi-dimensional liquid chromatography system, connected to a LTQ ion trap mass spectrometer. Relative quantitation of tryptic peptides was enabled through a label-free strategy. The unique protein ID and magnitude of differential expression was finally confirmed by a florescent Western blot methodology ECL Plex, enabling confirmation of both protein identity and physical properties as well as relative quantitation of potential cancer biomarkers.