Spatiotemporal and quantitative analyses of phosphoinositides - fluorescent probe-and mass spectrometry-based...

Comprehensive understanding of phosphoinositide signaling requires both spatiotemporal visualization and precise quantitative analysis of individual lipid species. Phosphoinositides, a family of phosphorylated derivatives of phosphatidylinositol (PI), are structurally diverse lipid messengers that orchestrate a wide range of cellular functions, including membrane trafficking, cytoskeletal dynamics, and signal transduction. Due to their dynamic metabolism and compartment-specific localization, their analysis demands complementary strategies that integrate live-cell imaging with molecular quantification. In this review, we first summarize the development and application of fluorescence-based probes designed to monitor the distribution and dynamics of phosphoinositides in living cells, highlighting their specificity, targeting mechanisms, and limitations. We then provide an overview of recent advances in mass spectrometry-based methodologies that enable high-sensitivity, isomer-resolved quantification of phosphoinositides in biological specimens, including improvements in lipid extraction, derivatization, and chromatographic separation. Together, these dual approaches offer synergistic insights into the biochemical and cellular regulation of phosphoinositide signaling.