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J Lipid Res. 2009 Jun;50(6):1229-36. doi: 10.1194/jlr.D800044-JLR200. Epub 2009 Jan 28.

Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles.

Author information

1
Section of Atherosclerosis and Vascular Medicine, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.

Abstract

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.

PMID:
19179308
PMCID:
PMC2681406
DOI:
10.1194/jlr.D800044-JLR200
[Indexed for MEDLINE]
Free PMC Article

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