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Analysis of oxidized apoprotein B-100 by mass spectrometry

Chulalongkorn University
Publication Date
  • Lipoproteins
  • Cholesterol
  • Oxidation
  • Mass Spectrometry
  • Apoprotein
  • Biology


Oxidatively modified LDL is likely to be the main source of cholesterol that accumulates in arteriosclerotic plaques. Trace amount of copper can induce LDL oxidation that generate the lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). These aldehydes especially HNE modify apoB-100 results in an increasing in net negative charge of apoB-100, leads to the decreased or completely lost recognition by LDL receptor. Leading to the accumulation of cholesterol or cholesterol esters in the arterial wall and formation of fatty streak in the arterial wall and served as the first stage of atherogenesis. In this research, LDL was prepared from EDTA plasma by sequential ultracentrifugation, and HNE/protein product was prepared from copper-mediated oxidized LDL and reduced with NaBH[subscript 4], following extraction of oxidized apoB-100 with CH[subscript 3]Cl/MeOH (2/1) and trypsin digestion. Later, the tryptic peptides were separated by reverse phase HPLC with ACN/H[subscript 2]O gradient elution. Finally, tandem mass spectrometry in precursor ion scanning of m/z 268 that correspond to the reduced form of the immonium ion of HNE-modified histidine, was used to determine the sites of modification on oxidized apoB-100. The modified peptides, T198 and T103 were found. Later the modified peptides were sequenced in product ion scanning mode to confirm the sites of modification. T198 and T103 with the sequence LH*VAGNLK and LLSGGNTLH*LVSTTK were presented (where * indicates adduction by HNE).

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