J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D700025-JLR200 on November 18, 2007

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Journal of Lipid Research, Vol. 49, 481-490, February 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr

Marcus Ståhlman{dagger}, Pia Davidsson*, Ida Kanmert*, Birgitta Rosengren*, Jan Borén{dagger}, Björn Fagerberg{dagger} and Germán Camejo1,*,{dagger}

* AstraZeneca R&D, Mölndal, Sweden
{dagger} Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska Academy, Gothenburg University, Sweden

Published, JLR Papers in Press, November 18, 2007.

1 To whom correspondence should be addressed. e-mail: german.camejo{at}astrazeneca.com

There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D2O) and sucrose. An advantage of the D2O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D2O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.

Supplementary key words deuterium oxide • differential ultracentrifugation • plasma • VLDL • LDL- and HDL-exchangeable apolipoproteins and proteins


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