Development of a 2-D apoB peptide profile to detect conformational changes associated with apoB-containing...

PTMs, such as glycosylation and phosphorylation of apolipoprotein B100 (apoB), are known to be involved with modulating the metabolism of apoB-containing lipoproteins. Current evidence suggests that intracellular and extracellular PTM of apoB are associated with various disorders such diabetes, dyslipidemia and atherosclerosis. The ability to identify and characterize the specific PTM of apoB correlating to specific pathologies may improve our understanding of the underlying molecular mechanisms regulating apoB metabolism. We have developed an assay to detect PTM and/or conformational changes in apoB isolated from the media of HepG2 cells. Using trypsin digestion in conjunction with 2-DE and Western blotting, a 2-D peptide fragment profile of apoB was established. The 2-D apoB profile was composed of a number of trypsin-generated fragments having a molecular mass between 10 and 188 kDa and a wide spectrum of isoelectric points. The 2-D apoB profile obtained from the media of HepG2 cells treated in the presence of agents (tunicamycin and glucosamine) known to modulate the PTM of apoB was distinct from that of control cells. Identifying changes in the 2-D apoB profile has the potential to not only provide insight into the underlying mechanisms regulating the metabolism of apoB-containing lipoproteins but may also have important implications for the development of novel diagnostic tools and/or future therapeutic agents.