onstrated for the analysis of lipid extracts from different biological materials ( 14-18 ), but the most promising advantage of the MALDI-TOF/MS technique turned out to be the possibility to perform lipid analysis avoiding extraction and/or separation steps.Recent advances in MALDI-TOF/MS techniques of lipid analysis have led to the direct coupling of , as well as to the direct analysis of tissue slices with the MALDI-MSI ( 23-25 ). In both cases, a plate containing the sample is coated with the matrix solution (silica plate or tissue on microscope glass plate, respectively), attached onto the MALDI target, and directly scanned with the laser to obtain a fast MS analysis of the lipids on the plate. Both these methods allow a fast and convenient lipid analysis, skipping extraction steps that are usually required for these kinds of analyses.In 2010 we developed a protocol that allows the direct lipid analysis by MALDI mass spectrometry in intact archaeal membranes without prior extraction/separation steps: lyophilized archaeal membranes were ground with 9-aminoacridine (9-AA) as dry matrix, and the powder mixture was crushed in a mechanical die press to form a thin pellet; small pieces of the pellet were then attached onto the MALDI target, and the archaeal membrane lipid profi le was directly achieved ( 26 ).Another new protocol for the direct lipid fi ngerprinting with MALDI-TOF/MS was published by Ferreira et al. in 2010 ( 27 ) Historically lipid profi ling of biological samples has been achieved with preliminary isolation of tissues, cells, membranes or organelles, followed by lipid extraction and purifi cation with chromatographic techniques, such as HPLC or (HP)TLC ( 1, 2 ), and by lipid characterization and/or quantitation with gas chromatography (GC) ( 3 ), GC-mass spectrometry (MS) ( 4 ), fast atom bombardment (FAB)-MS ( 5-7 ), , and/or NMR analyses ( 13 ).In the last decade, the capabilities of MALDI time-offl ight (TOF)/MS in lipid analysis have been largely dem-

The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

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