Serum proteins are often expressed as multiple isoforms with different isoelectric points (pI) and molecular masses. These diverse molecular forms result not only from genetic variations but also from post-translational modifications (PTM) such as phosophorylation, acetylation, glycosylation and oxidation. However, it is not known whether each isoform plays a distinct physiological role, nor whether differential expression of protein isoform correlates with a certain disease state. In order to investigate the relationship between protein modifications and diseases, a rapid detection system which enables us to distinguish modified from unmodified forms is critically important. We reported in the 4th JHUPO meeting a new isoelectric focusing chip in which micro-channels equipped with nano-pillar structure are fabricated on silicon substrates. Proteins electrophoretically-separated according to their pI's on the chip can be detected by subsequent MALDI mass spectrometry. Human apolipoprotein A1 (ApoA1), a major protein constituent of high density lipoproteins (HDL), is primarily synthesized and secreted by the liver. HDL executes retrograde transport of excess cholesterol from the peripheral tissues to the liver, thus playing an important role in regulating the level of cholesterol. ApoA1 has been shown to receive various PTMs, such as proteolysis, phosphorylation and oxidation. A recent report suggested that oxidation of specific amino acid residues in ApoA1 correlates with liver cancer. We are presently interested in the relationship between the expression profiles of ApoA1 isoforms and coronary heart disease, and have started to apply our chip system to investigate the expression profiles of ApoA1 isoforms. We have so far detected two ApoA1 species with different pI's in control serum from healthy persons. These results and the analysis of serum samples from patients will be presented in the meeting.
