Selective Enrichment\nvia TiO<sub>2</sub> Magnetic\nNanoparticles Enables Deep Profiling of Circulating Neutral Glycosphingolipids

Circulating neutral glycosphingolipids\n(neutral GSLs\n(nGSLs)) are\na unique subset of nGSLs that detach from organs or cell membranes\nand enter the bloodstream. Altered molecular distribution of circulating\nnGSL is increasingly associated with diseases. However, profiling\nof circulating nGSLs presents a lasting challenge due to their low\nabundances and structural complexity. Although TiO<sub>2</sub> magnetic\nnanoparticles (TiO<sub>2</sub> MNPs) were effective for the enrichment\nof nGSLs in brain tissue, the protocol showed limited selectivity\nfor circulating nGSLs because their abundances were 100-times lower\nin human plasma than in brain tissue. In this work, we optimized the\nkey parameters of selective enrichment by TiO<sub>2</sub> MNPs and\nachieved 1:10,000 selectivity for nGSLs over interfering phospholipids,\nwhile maintaining ∼70% recovery for different subclasses of\nnGSLs. By integrating TiO<sub>2</sub> MNP-based selective enrichment\nwith reversed-phase liquid chromatography mass spectrometry and charge-tagging\nPaternò-Büchi derivatization, we achieved deep profiling\nof over 300 structures of nGSLs and sulfatides across 5 orders of\nmagnitude in relative abundances, a significant leap regarding lipid\ncoverage. We also depicted the structural atlas of nGSLs with defined\nheadgroup, long-chain base, N-acyl chain, the location of desaturation,\nand 2-hydroxylation. Such information provides a valuable resource\nfor lipidomic studies concerning the roles of circulating nGSLs in\nhealth and diseases.