High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling

Abstract

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.