Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry

Introduction: Sphingolipids and glycosphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. They have also been identified for their role in pathology, where aberrant structures or abundances are observed.

The structural diversity and relatively low abundance of sphingolipids and glycosphingolipids within the overall lipid pool of a cell have made their comprehensive analysis challenging. For example, theoretical estimates predict that up to 200,000 unique glycosphingolipids molecules could exist given the 450 different glycan heads discovered thus far. The initial profiling and understanding of the glycosphingolipid molecules used analytical methodologies requiring the hydrolysis of the bond between the oligosaccharide and the sphingolipid but lacked information for the intact molecules.

In this study, we developed a robust and reproducible method for quantitatively profiling intact glycosphingolipids with automated compound identification. Our method employed nanoflow reverse-phase high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (nRP-HPLC-Q/ToF) to separate and detect glycosphingolipids from biological samples.