Abstract
A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.
Highlights
A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented
In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined
Quantification by nanoESI-MS/MS is achieved by comparing the ion intensities of the sulfated internal standard with the sulfated cholesterol in a precursor ion scan specific for sulfated ions (HSO4Ϫ) that give rise to a signal at m/z 97
Summary
A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. Protein and lipid transport between these organelles is established mainly by vesicles that bud off a donor membrane and fuse with an acceptor membrane. Such a difference can only be demonstrated by directly comparing the lipid compositions of both the vesicles and their donor membranes One candidate for such a carrier is the COPI (coat protein complex I)coated vesicle, available in highly purified form but at very low quantity [2]. The method is sensitive enough to allow the quantitative analysis of free cholesterol in as few as 250 Chinese hamster ovary (CHO) cells
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