Validation of Far Upstream Binding Protein (FUBP) Isoforms in Human Hepatocellular Carcinoma Samples Using MRM Initiated Detection and Sequencing (MIDAS) Approach

The over-expression of a novel protein family, far upstream binding proteins (FUBPs) was identified in human hepatocellular carcinoma (HCC) for the first time. They were obtained first by using two dimensional-difference gel electrophoresis (2D-DIGE.) coupled with mass spectrometry (MS). A complementary approach using a four-plex isobaric tagging off-line two dimensional liquid chromatography tandem mass spectrometry (4-plex iTRAQ off-line 2-D LC/MS/MS) was also conducted and these same FUBP isoforms were found to be regulated. Confirmation of these protein isoforms was first performed using western blots but this approach was dependent on the availability of antibodies against FUBP. Thus an alternative and faster approach using MIDAS methodology was also implemented to validate these protein isoforms. We report here that MIDAS could distinguish successfully the two FUBP isoforms in HCC, and at the same time provide quantification data which can be compared with all the quantification techniques employed here.