Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS

Abstract

By combining the partition method for enrichment of sulfatides without any chromatographic procedures and the preparation method of lysosulfatides, we succeeded in analyzing these sulfated glycosphingolipids from biological materials by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to reduce the complexity of mass fragmentation patterns within a day. We found that sulfated GalCer (HSO3-3Gal beta 1Cer) (SM4s [galactosylsulfatide]) was composed of different species. While composition of SM4s specifically depended on source materials, it always contained hydroxy fatty acids of various degrees. In addition to the common sphingoid 4-sphingenine (d18:1), uncommon/unusual sphingoids phytosphingosine (4-hydroxysphinganine) (t18:0), eicosasphinganine (d20:0), 4-eicosasphingenine (d20:1), and sphingadienine (d18:2) were easily detected. Finally, in addition to SM4s, sulfatide sulfated LacCer (HSO3-3Gal beta 4Glc beta 1Cer) (SM3 [sulfated lactosylceramide]) and sulfated Gg3Cer (GalNAc beta 4(HSO3-3)Gal beta 4Glc beta 1Cer) (SM2 [sulfated gangliotriaosylceramide]) were clearly detected in renal tubule cells. The major SM4s was composed of ceramides possessing d18:1 with C22 hydroxy fatty acids (C22:0 h), C23:0 h, and C24:0 h, whereas the major SM3/SM2 were composed of ceramides possessing t18:0 with C22 normal fatty acids (C22:0), C23:0, C24:0. Namely, in these two series of sulfatides, either fatty acids or sphingoids were hydroxylated, and chain lengths of these components were exactly the same, consequently resulting in a similar polarity of ceramide moieties in these sulfatide species. These results demonstrated diversities of sulfatide molecular species, not only with respect to sugar moieties but also to ceramide moieties, which are probably important for specific effective functions in particular microenvironments such as lipid membrane microdomains.

Quantitative Transcriptomic Profiling of Branching in a Glycosphingolipid Biosynthetic Pathway

“…GSL fractions equivalent to 1 mg of cellular protein were applied to a silica-based TLC plate and separated with a solvent (chloroform, methanol, 0.2% CaCl 2 , 60:35:8). Orcinol was used for the visualization of sugar moieties in GSLs and charring was carried out to gain sensitivity for the detection after orcinol staining (33). For detection of GM1, 670 ng/ml of biotinylated CTxB was overlaid and the signal was detected with Konica Immunostain HRP-1000 (Seikagaku).…”

Selective desorption/ionization of sulfatides by MALDI-MS facilitated using 9-aminoacridine as matrix

“…MALDI-MS, a widely used MS technology for analyses of protein and nucleotide polymers, possesses the potential for rapid analysis of sulfatides and has showed success in directly imaging and detecting sulfatide species from biological tissue samples (28)(29)(30)(31)(32). Quantitation of sulfatide species has previously been demonstrated by MALDI-MS after converting sulfatides to their lyso forms by saponifi cation and sample purifi cation using a C18 column or C18 tips ( 22,23 ).…”

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

“…Liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput [5,6,[12][13][14][15][16][17][18][19][20][21], but these reports did not fully characterize ceramide species including hydroxyl FAs (HFAs) and/or trihydroxy-LCBs (tLCBs) of both free ceramides and the constituent ceramides in GSLs. Namely, in the analyses of GSLs these investigations focused on sugar sequences by releasing sugars from the lipid portions [22,23]; therefore, detailed information regarding the constituent ceramide species in GSLs is limited [20].…”