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.
Highlights
Glycosphingolipids (GSL) are ubiquitous membrane components [1] predominantly found in the outer leaflet of the cell plasma membrane [2,3,4] of virtually all eukaryotic species [5] and some bacteria [6], where they are included in lipid rafts [1,5] together with cholesterol clusters, which serves as binding sites or receptors [4]
We have identified and structurally characterized 59 neutral (+14 species not confirmed by mass spectrometry (MS)/MS) and 64 acidic (+17 species not confirmed by MS/MS) GSL species together with 77 phospholipid species in human plasma (Figure 9). 37 of all 77 phospholipid species identified in human plasma were not confirmed by MS/MS, as phospholipids PE and LPE (35 lipid species in total) were not the principal subject of this study and do not interfere with any important glycosphingolipid subclass
The work reports the identification of 123 GSL species in the human plasma, including those not yet previously reported
Summary
Glycosphingolipids (GSL) are ubiquitous membrane components [1] predominantly found in the outer leaflet of the cell plasma membrane [2,3,4] of virtually all eukaryotic species [5] and some bacteria [6], where they are included in lipid rafts [1,5] together with cholesterol clusters, which serves as binding sites or receptors [4]. Lydic et al [37] came up with monophasic chloroform-methanol-water (1:2:0.74, v/v/v) solvent system facilitating the simultaneous analysis of highly polar and nonpolar lipids using the shotgun method This approach provided lipid profiles comparable to those obtained by well-established biphasic extraction methods, and enabled detailed lipidome analyses of broad range of GSL species without necessity for multi-steps extraction methods, where the partial redistribution between two phases is commonly inevitable [37,39]. Chemical derivatization strategies are frequently used to enhance the sensitivity for more accurate quantification [28] In respect of these facts, the utilization of monophasic water-soluble organic solvent systems seems to be the most favorable approach for the effective extraction of a wide range of GSL subclasses since some lipid species can be lost in the solvent system interface and/or their amounts may be decreased due to multi-steps extraction or redistribution between two phases when using traditional biphasic lipid extraction. GSL, such GasSLm, osnuocshiaalosdmihoenxoosiyallgoadnighleioxsoidsyelsg(aGnMgl3i)o, sairdeesca(lGleMd 3g)a,nagrleiocsaidllesd, ganadngtlhioeisrides, and their shorthand snhootarthioannhdansoatlaretiaodnyhbaesenaltrheoardoyubgheelyndthesocrroibuegdhilny oduerscprriebveidouins wouorrkpr[4e5v]i.ous work [45]
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