Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry.
Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w'') and fatty acyl chains (Y0'-w'') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.
Kaga
Naoko
N
Division of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan.
Kazuno
Saiko
S
Taka
Hikari
H
Iwabuchi
Kazuhisa
K
Murayama
Kimie
K
D000818:Animals; D002853:Chromatography, Liquid; D006358:Hot Temperature; D007790:Lactosylceramides / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D013058:Mass Spectrometry; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine
D007790:Lactosylceramides