Enrichment and MALDI-TOF-MS/MS analysis of phosphatidylinositol bisphosphates in brain tissue

Triazolium α-cyclodextrin click cluster-magnetic agarose bead conjugate (⁺CCC-MAB) was used to enrich phosphatidylinositol bisphosphates in brain tissue. The enriched sample was phosphate-methylated and analyzed by MALDI-TOF-MS/MS in positive ion mode. ⁺CCC-MAB effectively removed weak-binding interferences from the phosphoinositide extract and improved the signal-to-noise ratio. The MALDI-TOF-MS/MS fragment ion revealed sodium adducts of polar head groups, exhibiting a converse fragmentation pattern compared to LC-ESI fragmentation. Our ⁺CCC-MAB-based phosphoinositide enrichment method enabled MALDI-TOF-MS/MS to assign 38 peaks in brain tissue and identify two phosphatidylinositol monophosphates, fifteen bisphosphates, and two trisphosphates. To our knowledge, this is the first study to analyze phosphatidylinositol bisphosphates in brain tissue using specific PIP enrichment and phosphate-methylation with MALDI-TOF-MS/MS.