Derivatization strategy for sensitive identification of neutral and acidic glycosphingolipids using RPLC-MS

Glycosphingolipids are essential components of the cell membrane and implicate in a myriad of diseases. Glycosphingolipids are classified as neutral and acidic families based on the glycan structure. Neutral glycosphingolipids possess one or more neutral monosaccharide units, while acidic glycosphingolipids (such as gangliosides) contain one or more sialic acid (classified as NeuAc and NeuGc) linking on the glycan part. To better understand the metabolism process of glycosphingolipids, simultaneous identification of neutral and acidic glycosphingolipids is often required. However, it is hard to analyze gangliosides in positive-ion mode due to its poor ionization efficiency. Herein, we proposed a new strategy based on chemical derivatization to stabilize sialic acid and improve MS detection sensitivity of gangliosides in positive-ion mode. The carboxyl group of sialic acid were labeled with N,N-dimethylethylenediamine (DMEN), which contains a positively ionizable tertiary amine group. The labeling efficiency of DMEN was close to 100% and the ionization efficiency of gangliosides was enhanced for more than 4-fold. The fragments of DMEN labeled gangliosides provide a diagnosis ion to facilitate rapid structural assignments of gangliosides, and also help discrimination of isomeric gangliosides with different sialic acid location. By using DMEN derivatization, 45 glycosphingolipids were identified from human plasma, including 30 gangliosides and 15 neutral glycosphingolipids. Our strategy could therefore be a generic approach for simultaneous MS identification of neutral and acidic glycosphingolipids.