Brain phosphoinositide extraction, fractionation, and analysis by MALDI-TOF MS.
Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) can provide rapid, sensitive determinations of lipids from small tissue samples in both single determinations and automated high-throughput assays. MALDI-TOF MS is a sensitive, high-throughput technique for the determination of lipids such as the phosphoinositides, PtdIns (phosphatidylinositol), PIP (phosphatidylinositol-4-phosphate, and PIP2 (phosphatidylinositol-4,5-bisphosphate), but in crude extracts the signals are weak or not observed due in large part to ion suppression by phosphatidylcholine and other cationic lipids. A rapid separation step using a small column of a strong cation exchange (SCX) gel can be utilized easily and effectively to adsorb or capture cationic lipids from chloroform/methanol lipid extracts and provide substantially improved signals for the phosphoinositides. In this review, we describe the use of fractionation of a crude lipid extracts using cation exchange columns in conjunction with MALDI-TOF MS and appropriate internal standards to quantify the levels of phosphoinositides in small mammalian brain samples.
Johanson
Roy A
RA
Department of Neurology, Thomas Jefferson University Medical Center, Philadelphia, PA, USA.
Berry
Gerard T
GT
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D051379:Mice; D008810:Mice, Inbred C57BL; D010716:Phosphatidylinositols / Q000302:isolation & purification / Q000378:metabolism*; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002411:Cation Exchange Resins; D010716:Phosphatidylinositols