Comprehensive Identification of Glycosphingolipids in Human Plasma Using Hydrophilic Interaction Liquid Chromatography—Electrospray Ionization Mass Spectrometry

Abstract

Glycosphingolipids (GSL) represent a highly heterogeneous class of lipids with many cellular functions, implicated in a wide spectrum of human diseases. Their isolation, detection, and comprehensive structural analysis is a challenging task due to the structural diversity of GSL molecules. In this work, GSL subclasses are isolated from human plasma using an optimized monophasic ethanol–water solvent system capable to recover a broad range of GSL species. Obtained deproteinized plasma is subsequently purified and concentrated by C18-based solid-phase extraction (SPE). The hydrophilic interaction liquid chromatography coupled to electrospray ionization linear ion trap tandem mass spectrometry (HILIC-ESI-LIT-MS/MS) is used for GSL analysis in the human plasma extract. Our results provide an in-depth profiling and structural characterization of glycosphingolipid and some phospholipid subclasses identified in the human plasma based on their retention times and the interpretation of tandem mass spectra. The structural composition of particular lipid species is readily characterized based on the detailed interpretation of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) spectra and further confirmed by specific fragmentation behavior following predictable patterns, which yields to the unambiguous identification of 154 GSL species within 7 lipid subclasses and 77 phospholipids representing the highest number of GSL species ever reported in the human plasma. The developed HILIC-ESI-MS/MS method can be used for further clinical and biological research of GSL in the human blood or other biological samples.

Recommendations for good practice in MS-based lipidomics

“…Here, the RT should be used as an additional criterion for lipid species identification. Thus, for lipid class separation like HILIC, the observed RT is similar for the respective lipid class and follows polarity shifts, for example, by hydrocarbon chain length ( 37 , 77 ) or introduction of additional hydroxyl groups ( 111 ). In RP-HPLC, the separation is based on the hydrophobic interaction, which is related to the length of hydrocarbon chains, the number of double bonds, and other structural features, such as polar functional groups.…”

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

“…The ionization and fragmentation behavior of individual lipid classes is well known from previous works [4,12,24,[36][37][38][39]. Characteristic precursor and product ions (Table 1) were searched in both positive-ion and negative-ion ESI mass spectra for the identification of lipids in human plasma.…”

Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters

“…We used nano-HILIC MS/MS for the detection of neutral and acidic GSLs to curate a GSL database. 18 Statistic evaluation of nano-HILIC MS/MS data revealed significant differences between infected and noninfected samples. Subsequently, optimized AP-SMALDI MSI provided information about the topography of the GSL species in infected tissue.…”

“…We analyzed, as controls, the livers of hamsters infected by only female worms (no egg production) or noninfected. We used nano-HILIC MS/MS for the detection of neutral and acidic GSLs to curate a GSL database . Statistic evaluation of nano-HILIC MS/MS data revealed significant differences between infected and noninfected samples.…”