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标题：Ratio determination of plasma wild-type and L159R apoA-I using mass spectrometry: tools for studying apoA-IFin
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作者：Owen, John S. ; Bharadwaj, Manish S. ; Thomas, Michael J. 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2007
卷号：48
期号：1
页码：226-234
DOI：10.1194/jlr.D600031-JLR200
出版社：American Society for Biochemistry and Molecular Biology
摘要：In this report, methods are described to isolate milligram quantitiesof a mutant apolipoprotein A-I (apoA-I) protein for use in structure-functionstudies. Expression of the L159R apoA-I mutation in humans reducesthe concentration of plasma wild-type apoA-I, thus displayinga dominant negative phenotype in vivo. Earlier attempts to expressand isolate this mutant protein resulted in extensive degradationand protein misfolding. Using an Escherichia coli expressionsystem used predominantly for the isolation of soluble apoA-Imutant proteins, we describe the expression and purificationof L159R apoA-I (apoA-I_Fin) from inclusion bodies. In addition,we describe a mass spectrometric method for measuring the L159R-to-wild-typeapoA-I ratio in a 1 µl plasma sample. These new methodswill facilitate further studies into the mechanism behind thedominant negative phenotype associated with the expression ofthe 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