MS/MS ^ALL with SelexION®: A High‐throughput Lipidomic Solution for Untargeted Profiling

Global lipid profiling is a growing area of interest and is a primary means to measure the changes in lipid molecular species as a function of human disease or drug treatment. One of the most challenging aspects of lipid analysis by mass spectrometry is isobaric overlap, or molecules having the same or nearly the same mass. With direct infusion, or ‘Shotgun’ approaches, the user is able to rapidly profile biological samples. On the Sciex 6600+ TripleTOF®, the MSMS ALL method on this system generates data by collecting product ions of all precursor masses. Using this approach, thousands of lipids can be represented in a profile snapshot and allows for retrospective analysis of acquired data. However the issue of isobaric overlap is not addressed. Isobaric overlap is a complex problem in lipidomic workflows and can make positive identification or quantification of a lipid species very difficult. The issue of isobaric overlap is often approached with liquid chromatography separation. However, lengthy sample preparation, method development, and lengthy chromatographic run times for adequate separation can narrow the scope of the lipidome and diminish throughput. Differential ion mobility separation (DMS) is a relatively new ion mobility technology that uses a molecule’s dipole moment to isolate it rather than shape and size. The SelexION® device is installed at atmosphere, so the addition of chemical modifiers can be applied to the system to assist in additional separation. In this study, 1‐propanol is used as the chemical modifier to separate the six phospholipid subclasses while cardiolipin’s ‐2H charge state is used to separate this class without the assistance of chemical modifier. Unique compensation voltages are then found with standards and assigned to each lipid class to ensure lipid class isolation. In negative ion mode, all seven classes can be acquired in just under twelve minutes of total run time. This allows for untargeted, high‐throughput acquisition of the lipidome while simultaneously acquiring MS/MS spectra for lipid species identification. LipidView software is used to analyze the data collected from the SelexION® runs in a high throughput fashion. All samples from each class can be loaded and a lipid class can be assigned along with its corresponding COV. Since much of the isobaric overlap has been removed from the sample a positive identification and quantification can be made.

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