4.3. Multi-Dimensional MS-Based Shotgun Lipidomics The multi-dimensional MS (MDMS)-based shotgun lipidomics platform maximally exploits the unique chemistries inherent in distinct lipid classes to identify and quantify individual lipid species after direct infusion [4,10,48]. For example, MDMS-based

shotgun lipidomics utilizes a multiplexed extraction approach that exploits differential hydrophobicity or differential chemical stability under acidic or basic conditions to separate and/or enrich differential lipid classes by liquid/MLN8237 concentration liquid partitioning or by multiplexed chemistries [10]. MDMS-based shotgun lipidomics also exploits the differential charge Inhibitors,research,lifescience,medical properties to achieve selective ionization of differential Inhibitors,research,lifescience,medical lipid classes under multiplexed infusion conditions that allow intrasource separation of lipids in different classes or categories [49]. In addition, MDMS-based shotgun lipidomics exploits the uniqueness of individual lipid classes to identify and quantify lipids in specific lipid

classes. Examples include quantification Inhibitors,research,lifescience,medical of cardiolipins through use of the unique doubly-charged molecular ions resulting from the presence of two phosphate moieties present in cardiolipin resulting in M + 0.5 isotopologue patterns [50]; identification and quantification of phosphoethanolamine-containing lipid species Inhibitors,research,lifescience,medical by the specific derivatization of primary amine with fluorenylmethoxylcarbonyl (Fmoc) chloride [51]. MDMS-based shotgun lipidomics utilizes the principle of building block analysis for identification of individual lipid species by employing two powerful MS/MS techniques Inhibitors,research,lifescience,medical (i.e., PIS and NLS)

in a mass-ramp fashion [10]. Specifically, PIS or NLS of the fragment ion(s) resulting from the head group or the neutral loss of the head group building block identifies the lipid class of interest, and PIS or NLS of fatty acyl building blocks identifies the individual almost lipid species in the class. The class-specific diagnostic ions are also exploited for lipid quantification. In contrast to the other two shotgun lipidomics platforms, MDMS-based shotgun lipidomics quantifies the identified individual lipid species using a two-step procedure that incorporates not only exogenously added, pre-selected internal standards, but also endogenous lipid species that are quantifiable accurately in a full MS survey scan. Specifically, in the first step, the platform employs a full MS scan acquired after intrasource separation and the pre-selected internal standard of the class of interest for quantification of lipid species that are abundant and not overlapped with lipid species from other classes.