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Papers In Press, published online ahead of print June 1, 2009
J. Lipid Res., doi:10.1194/jlr.D800044-JLR200

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Journal of Lipid Research, Vol. 50, 1229-1236, June 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

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

John B. Massey^*, Henry J. Pownall^*, Stephen Macha, Jamie Morris, Matthew R. Tubb and R. A. Gangani D. Silva^1,

^* Section of Atherosclerosis and Vascular Medicine, Department of Medicine, Baylor College of Medicine, Houston, TX 77030
Department of Chemistry, Mass Spectrometry Services, University of Cincinnati, Cincinnati, OH 45221
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of a figure.

This work was supported by grants from the National Institutes of Health/NHLBI to R.A.G.D.S. (K99/R00, HL-1004925) and to H.J.P. (HL-30914 and HL-56865).

Published, JLR Papers in Press, January 28, 2009.

¹ To whom the correspondence should be addressed. e-mail: silvar{at}ucmail.edu

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.

Supplementary key words apolipoprotein A-I • phospholipids • cholesterol • matrix-assisted laser desorption/ionization mass spectrometry

Abbreviations: apoA-I, apolipoprotein A-I; apoA-IV, apolipoprotein A-IV; BS³, bis(sulfosuccinimidyl)suberate; DMPC, dimyristoyl-sn-glycero-3-phosphocholine; MS, mass spectrometry; M_r, molecular weight; PAGGE, polyacrylamide gradient gel electrophoresis; rHDL, reconstituted high density lipoprotein

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