Enrichment and MALDI-TOF MS Analysis of Phosphoinositides in Brain Tissue

Triazolium cyclodextrin click cluster (CCC) is an ideal scaffold to specifically bind phosphoinositides (PIPs) via multivalent electrostatic interaction. A new enrichment material, triazolium cyclodextrin click cluster-magnetic agarose bead conjugate (CCC-MAB), was synthesized and applied to the PIP enrichment of brain tissue. The enriched sample was analyzed using MALDI-TOF MS in negative ion mode without any derivatization. The PIP extract of brain tissue is known to contain abundant lipid interferences. By employing magnetic pull-down separation using CCC-MAB, we effectively removed the weak-binding interferences in the PIP extract, thereby improving the signal-to-noise ratio (S/N) of the PIPs. Our CCC-MAB-based PIP enrichment enabled us to analyze 16 PIP species in brain tissue. Six species with high S/N were assigned by MS/MS, while the remaining 10 species with low S/N were characterized by an empirical selection guide based on the biological relevance of PIPs. We conclude that CCC-MAB-based PIP enrichment is a promising MALDI sample preparation method for specific PIP analysis in brain tissue.