Shotgun analysis of phospholipids from mouse liver and brain by nanoflow liquid chromatography/tandem mass spectrometry.

It was demonstrated that a shotgun approach can be utilized for the characterization of phospholipids (PLs) extracted from mouse liver and brain by using nanoflow reversed phase liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). In this study, a dual scan method was introduced for the high throughput analysis of complex PL mixtures. Two consecutive LC-ESI-MS-MS runs were made in positive ion mode (for phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs)) first followed by analysis in negative ion mode (for phosphatidylserine (PSs) and phosphatidylinositol (PIs)) using the same binary gradient elution with and without adding formic acid, respectively. The separation of the PLs was carried out using a home made pull tip capillary column (C18) with an end frit. The MS analysis of the eluted PL molecules was performed with a precursor scan followed by a data dependent MS-MS scan. The developed dual scan method was tested with the extracts of PCs and PIs mixtures from soybean, PEs from Escherichia coli, and PSs from bovine brain. It was further applied for the characterization of intact PL samples that were extracted from both mouse liver and mouse brain in the laboratory, and resulted in the identification of 90 and 80 PL species, respectively.