Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.
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.
Sandhoff
R
R
Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Brügger
B
B
Jeckel
D
D
Lehmann
W D
WD
Wieland
F T
FT
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000032:analysis* / Q000592:standards; D006224:Cricetinae; D004875:Ergosterol / Q000032:analysis; D005453:Fluorescence; D006056:Golgi Apparatus / Q000737:chemistry; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D008832:Microchemistry; D010743:Phospholipids / Q000032:analysis; D011725:Pyridines; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013265:Stigmasterol / Q000032:analysis; D013461:Sulfur Oxides
D007202:Indicators and Reagents; D010743:Phospholipids; D011725:Pyridines; D013461:Sulfur Oxides; D002784:Cholesterol; D013265:Stigmasterol; C011118:sulfur trioxide; C023666:pyridine; D004875:Ergosterol