Selective Enrichment via TiO₂ Magnetic Nanoparticles Enables Deep Profiling of Circulating Neutral Glycosphingolipids

Circulating neutral glycosphingolipids (neutral GSLs (nGSLs)) are a unique subset of nGSLs that detach from organs or cell membranes and enter the bloodstream. Altered molecular distribution of circulating nGSL is increasingly associated with diseases. However, profiling of circulating nGSLs presents a lasting challenge due to their low abundances and structural complexity. Although TiO₂ magnetic nanoparticles (TiO₂ MNPs) were effective for the enrichment of nGSLs in brain tissue, the protocol showed limited selectivity for circulating nGSLs because their abundances were 100-times lower in human plasma than in brain tissue. In this work, we optimized the key parameters of selective enrichment by TiO₂ MNPs and achieved 1:10,000 selectivity for nGSLs over interfering phospholipids, while maintaining ∼70% recovery for different subclasses of nGSLs. By integrating TiO₂ MNP-based selective enrichment with reversed-phase liquid chromatography mass spectrometry and charge-tagging Paternò-Büchi derivatization, we achieved deep profiling of over 300 structures of nGSLs and sulfatides across 5 orders of magnitude in relative abundances, a significant leap regarding lipid coverage. We also depicted the structural atlas of nGSLs with defined headgroup, long-chain base, N-acyl chain, the location of desaturation, and 2-hydroxylation. Such information provides a valuable resource for lipidomic studies concerning the roles of circulating nGSLs in health and diseases.