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No. 6 (PC 6)
Analysis of cholesteryl ester hydroperoxides in oxidized lipoprotein by Use of an
Orbitrap LC-MS
Shu-Ping Hui 1 , Toshihiro Sakurai 1 , Futaba Ohkawa 1 , Hiroaki Furumaki 1 , Shigeki Jin 1 , Hirotoshi
Fuda 1 , Seiji Takeda 1 , Takao Kurosawa 2 , Hitoshi Chiba 1
1 2
Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan Faculty
of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido
061-0293, Japan
Oxidation of cholesteryl esters in lipoproteins by reactive oxygen species yields cholesteryl ester
hydroperoxides (CEOOH). In this study, we developed a novel method for identification and
characterization of CEOOH molecules in human lipoproteins by use of reversed-phase liquid
chromatography with an hybrid linear ion trap–Orbitrap mass spectrometer (LC–LTQ Orbitrap).
Electrospray ionization tandem mass spectrometric analysis was performed in both positive-ion and
negative-ion modes. Identification of CEOOH molecules was completed by use of high-massaccuracy
(MA) mass spectrometric data obtained by using the spectrometer in Fourier-transform (FT) mode.
Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy
donor were oxidized by CuSO4, furnishing oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No
CEOOH molecules were detected in the nLDL and the nHDL, whereas six CEOOH molecules were
detected in the oxLDL and the oxHDL. In positive-ion mode, CEOOH was detected as [M + NH4] + and
[M + Na] + ions. In negative-ion mode, CEOOH was detected as [M +CH3COO] − ions.
CEOOH were more easily ionized in positive-ion mode than in negative-ion mode. The LC–LTQ
Orbitrap method was applied to human plasma and six species of CEOOH were detected. The limit of
detection was 0.1 pmol (S/N=5:1) for synthesized CEOOH.
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