Detection, imaging and quantification of phosphoinositides using ion chromatography suppressed conductivity and mass...

Phosphoinositide lipids have been implicated in aging, health and disease. Prior studies have identified enzymes critical for lipid metabolism, such as synthetic and degrading enzymes, which have different specificities based on the composition of the inositol ring as well as specific sub-cellular localization and multiple regulatory pathways. In addition to understanding the regulation of enzyme expression, activity and localization, it is critical to investigate levels of the specific lipids of interest. Current methods for detection and quantification of lipids include high performance liquid chromatography/ion chromatography (HPLC/HPIC) suppressed conductivity detection and mass spectrometry. Isobaric polyphosphorylated lipid species can be differentially separated using HPIC methods based on the differential chemical properties of the phosphorylation sites on the inositol ring, which are not able to be detected by mass spectrometry. However, mass spectrometry has the advantage of detecting the acyl chain composition for the individual species which has been shown to affect the signaling properties of the lipids. Emerging methods using mass spectrometry imaging offer in situ localization of the specific lipids within tissue but do not yet achieve single cell or intracellular spatial resolution. We describe methods for detection of polyphosphorylated phosphoinositides using HPIC and both untargeted and targeted imaging mass spectrometry using matrix-free desorption electrospray ionization. We also provide considerations for optimization of the methods as well as quantification of the resulting high dimensional data. Based on the central role phosphoinositides play in neuronal function and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Down Syndrome, and Traumatic Brain Injury, sensitive and accurate detection and quantification is paramount for mechanistic studies as well as biomarker and therapeutic development.