Advanced lipidomic techniques for high-throughput profiling of complex sphingolipids in plant tissues

Sphingolipids are essential membrane components that regulate various cellular functions by forming ordered domains. The structural core of sphingolipids comprises a long-chain sphingoid base and its N-acylated ceramide, modified with diverse head groups to form complex sphingolipids. In plants, sphingolipid structures exhibit unique structural diversity in both the ceramide moiety and head group. The complicated sphingolipid structures suggest unique functions, but complicate lipidomic studies. A new methodology was established for simple sample preparation and targeted mass spectrometry, to advance high-throughput techniques for plant sphingolipidomics. A single-tube extraction protocol, consisting of the sequential procedures of enzyme inactivation, alkaline saponification and acidic 1-butanol phase partitioning, was optimized to enable rapid (~ 2.5 h) preparation of all sphingolipid classes with improved recovery. Over 900 molecular species from a theoretical plant sphingolipidome library were separated via reverse-phase liquid chromatography and quantified in a single run using scheduled monitoring of targeted mass spectrometry. This method enables large-scale profiling of the natural distribution of sphingolipids across diverse plant materials.