Phenotyping apolipoprotein E*3-Leiden transgenic mice by two-dimensional polyacrylamide gel electrophoresis and mass spectrometric identification

Apolipoprotein E (ApoE) plays an important role in cholesterol and triglyce ride metabolism, being one of the major structural components of chylomicro ns and very low density lipoprotein (VLDL) remnants. ApoE functions as a li gand in the receptor-mediated uptake of these remnants from the blood by th e liver. A variant form of ApoE, apolipoprotein E*3-Leiden, shows reduced a ffinity for the low density lipoprotein (LDL) receptor, and results in the dominant expression of type III hyperlipoproteinemia. Two-dimensional elect rophoresis (2-DE) has been used to characterise protein expression in serum samples from control and transgenic mice expressing the human ApoE*3-Leide n mutation, fed a cholesterol-rich diet,and transgenic mice fed a normal di et. For the identification of proteins, single silver-stained spots were ex cised from the 2-DE gels are subjected to in-gel enzymatic digestion. Extra cted peptides were analysed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This proteomic approach ha s enabled the ApoE*3-Leiden variant to be positioned in a 2-DE separation o f serum proteins, and has identified changes in the expression of haptoglob in, indicating that this protein may provide a marker for the potential ons et of atherosclerosis.