Strategy for Comprehensive Profiling and Identification of Acidic Glycosphingolipids Using Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Acidic glycosphingolipids (AGSLs),
which mainly consist of ganglioside
and sulfatide moieties, are highly concentrated in the central nervous
system. Comprehensive profiling of AGSLs has historically been challenging
because of their high complexity and the lack of standards. In this
study, a novel strategy was developed to comprehensively profile AGSLs
using ultra-high-performance liquid chromatography–quadrupole
time-of-flight mass spectrometry. Ganglioside isomers with different
glycan chains such as GD1a/GD1b were completely separated on a C18
column for the first time to our knowledge, facilitated by the addition
of formic acid in the mobile phase. A mathematical model was established
to predict the retention times (RTs) of all theoretically possible
AGSLs on the basis of the good logarithmic relationship between the
ceramide carbon numbers of the AGSLs in the reference material and
their RTs. A data set was created of 571 theoretically possible AGSLs,
including the ceramide carbon numbers, RTs, and high-resolution quasi-molecular
ions. A novel fast identification strategy was established for global
AGSL profiling by comparing the high-resolution quasi-molecular ions
and RTs of the tested peaks to those in the data set of 571 AGSLs.
Using this strategy, 199 AGSL candidates were identified in rat brain
tissue. MS/MS fragments were further collected for these 199 candidates
to confirm their identity as AGSLs. This novel strategy was employed
to profile AGSLs in brain tissue samples from control rats and model
rats with bilateral common carotid artery (2-VO) cerebral ischemia.
Forty AGSLs were significantly different between the control and model
groups, and these differences were further interpreted.