Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

Abstract

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated (3,5)A ions, indicating Hex 1-4 Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.

Alteration of fatty acid molecular species in ceramide and glucosylceramide under heat stress and expression of sphingolipid-related genes

“…MS/MS analysis of m/z 560 with high-energy CID fragmentation showed typical fragments m/z 264 and m/z 376 derived from d18:1, and m/z 304 from C16:0 (Figure 3(c)) [25]. (Figure 3(d)) [25].…”

“…MS/MS analysis of m/z 560 with high-energy CID fragmentation showed typical fragments m/z 264 and m/z 376 derived from d18:1, and m/z 304 from C16:0 (Figure 3(c)) [25]. (Figure 3(d)) [25]. While it was hard to detect m/z 414 derived from C24:1, molecular ion m/z 670 was speculated as d18:1 -C24:1 from the metabolic relationship.…”

Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells

“…Sphingolipids were purified from 5×10 7 cells as previously described [3,4]. Briefly, lipids were extracted with a chloroformmethanol mixture.…”

“…Mass spectrometry was performed as previously described [3,4]. Lipids were prepared as 10-350 pmol/μl solutions in chloroform:methanol (2:1).…”

“…All mass spectrometric data were acquired and analyzed using MALDI-MS software (Shimadzu/Kratos, UK). The compositions of molecular species from each class of lipid were calculated from the peak areas obtained from spectra as previously described [3,4].…”