Calditocaldarchaeol (neutral tetraether lipid) from Sulfolobus acidocaldarius (acidothermophilic archaea) and intact total lipid from the thermoacidophilic archaea Sulfolobus sp. was examined by electrospray ionization time-of-flight mass spectrometry in the negative-ion mode using high resolution. When the sample was injected as a solution in a 3:1 mixture of methanol (MeOH) and chloroform (CHCl3) using an infusion system, the total ether lipid afforded molecular-related ions as [M - H]- for acidic polar lipids containing a phosphoric or sulfuric group, and as [M + Cl]- ion for neutral glycolipids. The attachment of chloride was confirmed by the observation of [M + Br]- ion, instead of [M + Cl]- ion, when a 3:1 mixture of MeOH and CHBr3 was used in place of MeOH-CHCl3 as the solvent. The composition of tetraether neutral glycolipids that are different from each other only in the number of five-membered rings in the isoprenoid chain was determined on the basis of the isotope-resolved mass spectrum of [M + Cl]- ions. As for acidic tetraether lipids, molecular-related ions [M - H]-) were not observed when the 3:1 MeOH-CHBr3 mixture was used as the solvent. These results together afforded a facile method of distinguishing neutral from acidic tetraether lipids in intact total lipids of acidothermophilic archaea. This method was applied to determine the difference of the number of five-membered rings in isoprenyl chains of neutral tetraether glycolipids yielded by the Sulfolobus sp. grown at different temperatures. Discrimination of neutral tetraether glycolipids from acidic tetraether lipids in the total lipids obtained from Thermoplasma sp. was also achieved by this method.
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