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Journal of Lipid Research, Vol. 48, 226-234, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-I_Fin

John S. Owen^*, Manish S. Bharadwaj, Michael J. Thomas^*, Shaila Bhat, Michael P. Samuel^* and Mary G. Sorci-Thomas^1,*,

^* Department of Biochemistry, Center for Lipid Science, Wake Forest University Medical Center, Winston-Salem, NC 27101
Department of Pathology, Center for Lipid Science, Wake Forest University Medical Center, Winston-Salem, NC 27101

Published, JLR Papers in Press, October 28, 2006.

¹ To whom correspondence should be addressed. e-mail: msthomas{at}wfubmc.edu

In this report, methods are described to isolate milligram quantities of a mutant apolipoprotein A-I (apoA-I) protein for use in structure-function studies. Expression of the L159R apoA-I mutation in humans reduces the concentration of plasma wild-type apoA-I, thus displaying a dominant negative phenotype in vivo. Earlier attempts to express and isolate this mutant protein resulted in extensive degradation and protein misfolding. Using an Escherichia coli expression system used predominantly for the isolation of soluble apoA-I mutant proteins, we describe the expression and purification of L159R apoA-I (apoA-I_Fin) from inclusion bodies. In addition, we describe a mass spectrometric method for measuring the L159R-to-wild-type apoA-I ratio in a 1 µl plasma sample. These new methods will facilitate further studies into the mechanism behind the dominant negative phenotype associated with the expression of the L159R apoA-I protein in humans.

Supplementary key words apolipoprotein A-I • protein expression • inclusion bodies • apolipoprotein A-I mutant • high-performance liquid chromatography-electrospray mass spectrometry • high-performance liquid chromatography-electrospray tandem mass spectrometry • apolipoprotein A-I_Fin • high density lipoprotein • mass spectrometry • L159R apolipoprotein A-I

Abbreviations: apoA-I, apolipoprotein A-I; CCB, chitin column buffer; IPTG, isopropylthio-ß-D-galactoside; LC-MS, high-performance liquid chromatography-electrospray mass spectrometry; LC-MS/MS, high-performance liquid chromatography-electrospray tandem mass spectrometry; LDLr^–/–, apoA-I^–/–, low density lipoprotein receptor-deficient/apolipoprotein A-I-deficient

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