Ether Lipids

Hydrobiology of Sambhar Salt Lake a Ramsar site, Rajasthan was studied with respect to their chemical composition of brine and extremely haloalkaliphililes. Chemical compositions of brine samples were analyzed. Brine had pH value of 11 and a total salt content 30% (w/v). The sodium chloride and sodium bicarbonate were principal salts of brine. Metals like Fe, Mn, Zn, Cu, As, Cr, Pb, and Cd were detected in significant amount. Total 64 morphologically distinct isolates were recovered from brine samples out of that five were confirmed as red extremely haloalkaliphilic archaeabacteria and identified by using 16S rRNA sequencing method. All five isolates were required high salt (10-30%) and alkaline pH (9-10) for their optimum growth and pigmentation.16S rRNA sequencing revealed that selected isolates were members of Natronococcus geneus.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

This report describes the synthesis of reversed structured 1-O-alkyl-2,3-diacylsn-glycerols (DAGEs) possessing a pure saturated even number fatty acid (C6:0-C16:0) at the sn-2 position along with a pure EPA or DHA located at the terminal sn-3 position of the glycerol backbone of chimyl, batyl and selachyl alcohols. These adducts were synthesized by a highly efficient two-step chemoenzymatic process involving an immobilized Candida antarctica lipase to introduce pure EPA and DHA activated as oxime esters exclusively to the sn-3 terminal position of enantiopure chimyl, batyl and selachyl alcohols in excellent yields. The saturated fatty acids were subsequently incorporated to the remaining sn-2 position of the resulting 3-monoacylglyceryl ethers (3-MAGEs) using EDAC coupling agent in the presence of DMAP in very high to excellent yields (85%-98%). No losses of enantiomeric composition were observed during these processes. The multiple utilities of the resulting focused library of reversed structured DAGEs are discussed including how such compounds may possibly be utilized within the pharmaceutical area.

In aggressive tumors, alkylglyceronephosphate synthase (AGPS) controls cellular ether phospholipid utilization and metabolism to promote cancer cell proliferation and motility. SAR studies on the first-in-class AGPS inhibitor 1, discovered by our group, led to the 2,6-difluoro analog 2i which showed higher binding affinity than 1 in vitro. In 231MFP cancer cells, 2i reduced ether lipids levels and cell migration rate. When tested in PC-3 and MDA-MB-231 cancer cells, 2i specifically impaired epithelial to mesenchymal transition (EMT) by modulating E-cadherin, Snail and MMP2 expression levels. Moreover, the combination of siRNAs against AGPS and 2i provided no additive effect, confirming that the modulation of 2i on EMT specifically relies on AGPS inhibition. Finally, this compound also affected cancer cell proliferation especially in MDA-MB-231 cells expressing higher AGPS level, whereas it provided negligible effects on MeT5A, a non-tumorigenic cell line, thus showing cancer specificity.

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Alkylglycerols (alkyl-Gro) are ether lipids abundant in the liver of some elasmobranch fish species such as ratfishes and some sharks. Shark liver oil from Centrophorus squamosus (SLO), or alkyl-Gro mix from this source, have several in vivo biological activities including stimulation of hematopoiesis and immunological defences, sperm quality improvement, or anti-tumor and anti-metastasis activities. Several mechanisms are suggested for these multiple activities, resulting from incorporation of alkyl-Gro into membrane phospholipids, and lipid signaling interactions. Natural alkyl-Gro mix from SLO contains several alkyl-Gro, varying by chain length and unsaturation. Six prominent constituents of natural alkyl-Gro mix, namely 12:0, 14:0, 16:0, 18:0, 16:1 n-7, and 18:1 n-9 alkyl-Gro, were synthesized and tested for anti-tumor and anti-metastatic activities on a model of grafted tumor in mice (3LL cells). 16:1 and 18:1 alkyl-Gro showed strong activity in reducing lung metastasis number, while saturated alkyl-Gro had weaker (16:0) or no (12:0, 14:0, 18:0) effect. Multiple compounds and mechanisms are probably involved in the multiple activities of natural alkyl-Gro.

Alkylglycerols (alkyl-Gro) are ether lipids abundant in shark liver oil (SLO), and oral SLO or alkyl-Gro mix from this source have several in vivo biological activities including stimulation of haematopoiesis an immunological defences, or anti-tumour and anti-metastasis activities in vivo. Composition of natural alkyl-Gro mix contains several alkyl-Gro varying by chain length and unsaturation, and individual antitumour activity of each molecule present in natural mix remained unknown. We synthesized six prominent constituents of natural alkyl-Gro mix, namely 12:0, 14:0 16:0, 18:0, 16:1 n-7, and 18:1 n-9 alkyl-Gro. Using an in vivo model of grafted tumour in mice (3LL cells), we studied and compared the oral anti-tumour and anti-metastasis activities of each of these 6 alkyl-Gro. 16:1 and 18:1 alkyl-Gro showed strong activity in reducing lung metastasis number, while saturated alkyl-Gro had weaker (16:0) or no (12:0, 14:0, 18:0) effect. Spleen weights at day 20 after graft were also measured and showed tremendous variations depending on the treatment. Tumour graft resulted in a raise in spleen weight in control group, this raise was nearly abolished in 16:1 and 18:1 alkyl-Gro-treated mice, and was reduced in 14:0 and 16:0 alkyl-Gro-treated mice. Conversely, 18:0 alkyl-Gro-treated mice showed spleen weigh raise as compared with untreated grafted mice. These new data demonstrate a prominent role of unsaturation in the anti-tumour activities of alkyl-Gro.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

In aggressive tumors, alkylglyceronephosphate synthase (AGPS) controls cellular ether phospholipid utilization and metabolism to promote cancer cell proliferation and motility. SAR studies on the first-in-class AGPS inhibitor 1, discovered by our group, led to the 2,6-difluoro analog 2i which showed higher binding affinity than 1 in vitro. In 231MFP cancer cells, 2i reduced ether lipids levels and cell migration rate. When tested in PC-3 and MDA-MB-231 cancer cells, 2i specifically impaired epithelial to mesenchymal transition (EMT) by modulating E-cadherin, Snail and MMP2 expression levels. Moreover, the combination of siRNAs against AGPS and 2i provided no additive effect, confirming that the modulation of 2i on EMT specifically relies on AGPS inhibition. Finally, this compound also affected cancer cell proliferation especially in MDA-MB-231 cells expressing higher AGPS level, whereas it provided negligible effects on MeT5A, a non-tumorigenic cell line, thus showing cancer specificity.

The surface behavior of six different ether lipids from archaebacteria, based on condensation of glycerol or more complex polyols with two isoprenoid alcohols at 20 or 40 carbon atoms, was investigated in monolayers at the air-water interface. The compounds with no complex polar group (GD, GDGT, GDNT) form monolayers showing a reversible collapse at surface pressure as low as 22 dynes/cm. This collapse pressure decreases with temperature in such a way that the film tension remains constant. In condensed films, these molecules do not assume a completely upright position. Lipids with complex polar ends (HL, GLB, PLII) form films more stable to compression. Forcearea characteristics and surface moment values of HL monolayers are similar to those of analogous ester lipids with fatty acid chains. Monolayers of the two bipolar lipids, GLB and PLII, at room temperature present a more condensed state, probably due to the lateral cohesion between long alkyl chains, but a lower collapse pressure. For all bipolar lipids, the area expansion induced by temperature increase is larger than that of monopolar ones.

Lipase-catalyzed ethanolysis of squalene free shark liver oil has been investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to ten different lipases were compared. The ratio oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed although poor discrimination against diacyl glycerol ether was attained. In this study, the best results in terms of selectivity and stability were obtained with immobilized lipase from Candida antarctica (Novozym 435). Ethanolysis reaction in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacyl glycerol ethers and monoacylglycerol ethers.

erophospholipids, allowing a rapid, sensitive, and highly informative approach for the direct analysis of cell lipid extracts ( 2 ). Notably the use of the novel matrix 9-aminoacridine in MALDI-TOF/MS enables the fast and reliable detection of anionic phospholipids and also cardiolipin species ( 2 ). In our study we extended the use of 9-aminoacridine as a matrix for the MALDI-TOF/MS to glycerolipids and glycerophospholipids of extremely halophilic microorganisms belonging to the Archaea kingdom. The extremely halophilic archaeons are well adapted to life in NaClsaturated aquatic ecosystems, where they are exposed to extremely low water activity and desiccation stress. Glycerolipids and glycerophospholipids are defi ned as glycerol and glycerol-phosphate derivatives, respectively ( 3 ). Whereas in bacteria and eukaryotic cells, glycerolipids and glycerophospholipids are constituted by straight-chain fatty acids linked to glycerol by ester bonds, in the archaeal microorganisms, the hydrophobic chains are isopranoid chain alcohols linked to glycerol by ether bonds ( 4 ). The stereochemistry of archaebacterial membrane lipids is also different from that of other membrane lipids. In archaebacterial lipids, the glycerol group is alkylated at the sn -2 and sn -3 positions, at variance from bacterial and eukaryotic lipids, whose chains are linked at the sn -1 and sn -2 positions ( 4 ). The ether bond and the absence of unsaturations in the hydrophobic tail of the phospholipids (and other polar lipids present in archaeal membranes) Abstract A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacterium salinarum was selected as model membrane. Lyophilized purple membrane were grinded 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 to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholipids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix. -Angelini, R., F. Babudri, S. Lobasso, and A. Corcelli. MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine.

Lipids from the extremely halophilic Archaea, Haloarcula marismortui, contain abundant phytanyl diether phospholipids, namely archaetidic acid (AA), archaetidylglycerol (AG), archaetidylglycerosulfate (AGS), with mainly archaetidylglycerophosphate methyl ester (AGP-Me). These were accompanied by a triglycosyl archaeol (TGA), lacking characteristic sulfate groups. Tandem-mass spectrometry was employed to provide fingerprints for identifying these known lipids, as well as small amounts of unsaturated phospholipids. These contained 3 and 6 double bonds in their archaeol moiety, suggested by negative tandem-MS of intact phospholipids, as indicated by differences between their pseudo-molecular ion and specific fragment ions designated as p 2. The core ether lipids were confirmed by electrospray ionization mass spectrometry (ESI-MS) as 2,3-di-O-phytanylsn-glycerol (C20, C20), which gave rise to a precursor-ion at m/z 660 [M1Li] 1 , and its fragment ion at m/z 379 [M1Li] 1 , consistent with mono-O-phytanyl-glycerol. Furthermore, lithiated ions at m/z 654 (MS 1), 379 (MS 2) and m/z 648 (MS 1), 373 (MS 2), combined with 1 H/ 13 C NMR chemical shifts at d 5.31-121.6 (C2/2′-H2/2′), 5.08-124.9 (C6/6′-H6/6′) and 5.10-126.0 (10/10′-H10/10′) confirmed the presence of unsaturated homologs of archaeol. We carried out a comprehensive study on the lipids present in cells of H. marismortui. We used positive and negative ESI-MS with tandem-MS, which served as a fingerprint analysis for identifying the majority of component lipids.-de Souza, L.

Scalloped hammerhead shark (Sphyrna lewini) is a top predator on the ocean that has important ecological and economical values. Unfortunately, juvenile hammerhead sharks are commonly caught as an unintentional catch in Aceh Jaya waters. This study was conducted to provide information on critical habitat and fisheries characteristic of endangered scalloped hammerhead shark as a basis for management and protection of shark and ray in Aceh Jaya. The study was conducted from July to September and divided into habitat survey and landing monitoring. In the habitat trip survey, the observer joined in fishing boats to observe and record shark captures locations, meanwhile, the landing survey is conducted by collecting landing data on boats in the Rigaih fishing harbour, Aceh Jaya. We recorded 11 individuals from 25 trip of habitat survey and 142 individuals from 29 boat trips in landing monitoring. All scalloped hammerhead sharks we recorded in the study are juvenile with the average total length of 63 cm for habitat survey and 68 cm for landing monitoring. Fishing ground is recorded within 12 miles from the shore of Aceh Jaya. Most of the fleets are small boats below 5 GT with gillnets as their fishing gears targeting reef fish, shrimps, and small pelagic fish. This results in the additional observation of ecosystem characteristic suggested that the coastal waters of Aceh Jaya is a suitable for hammerhead shark critical habitat.

Tonic immobility is an induce method to achieve a temporary condition of cataleptic-like, which is a sudden loss of muscle tone, as a result of various procedures such as situate the object in an "unnatural" position or situation. This method has been widely applied on elasmobranch species, except for species of Hemiscyllidae. This study was intended to assess the application of tonic immobility on Hemiscyllium halmahera-an endemic shark to Halmahera Islands, Indonesia. Our study has shown that the average induction time ranged from 11.49 to 40.12 seconds, while the hypnosis duration ranged from 6.51 to 28.36 seconds. Based on our results, tonic immobility can be applied as non-lethal method to Hemiscyllium halmahera during capture and physical examination such as morphometric and weight measurement. However, we suggested to investigate further physiology examination such as heart rate, and breathing frequency. during tonic immobility phase on Halmahera walking shark or other reef shark species.

The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thinlayer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the threelaminar structure of the cell wall, which differs significantly from that of other Haloarchaea.

This article is available online at http://www.jlr.org erophospholipids, allowing a rapid, sensitive, and highly informative approach for the direct analysis of cell lipid extracts (2). Notably the use of the novel matrix 9-amino-acridine in MALDI-TOF/MS enables the fast and reliable detection of anionic phospholipids and also cardiolipin species (2). In our study we extended the use of 9-aminoacridine as a matrix for the MALDI-TOF/MS to glycerolipids and glycerophospholipids of extremely halophilic microorganisms belonging to the Archaea kingdom. The extremely halophilic archaeons are well adapted to life in NaCl-saturated aquatic ecosystems, where they are exposed to extremely low water activity and desiccation stress. Glycerolipids and glycerophospholipids are defi ned as glycerol and glycerol-phosphate derivatives, respectively (3). Whereas in bacteria and eukaryotic cells, glycerolipids and glycerophospholipids are constituted by straight-chain fatty acids linked to glycerol by ester bonds, in the archaeal microorganisms, the hydrophobic chains are isopranoid chain alcohols linked to glycerol by ether bonds (4). The stereochemistry of archaebacterial membrane lip-ids is also different from that of other membrane lipids. In archaebacterial lipids, the glycerol group is alkylated at the sn-2 and sn-3 positions, at variance from bacterial and eukaryotic lipids, whose chains are linked at the sn-1 and sn-2 positions (4). The ether bond and the absence of unsaturations in the hydrophobic tail of the phospholip-ids (and other polar lipids present in archaeal membranes) Abstract A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extrac-tion/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacte-rium salinarum was selected as model membrane. Lyophilized purple membrane were grinded 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 to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholip-ids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix. — Angelini, R., F. Babudri, S. Lobasso, and A. Corcelli. MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine. J. Lipid Res. 2010. 51: 2818–2825. Supplementary key words glycerolipids (diphytanylglycerol ether an-alogs) • glycerophospholipids (diphytanylglycerolphosphate ether ana-logs) • Halobacterium salinarum • purple membrane • matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry Today shotgun ESI-MS lipidomics represents one of the most powerful tools of analysis of the total lipid extract of biomembranes, allowing the detection of both zwitterionic and acidic phospholipids (1). Recently, a matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF/MS) approach , based on the use of 9-aminoacridine (9-AA) as matrix, has been developed for the rapid analysis of glyc

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyti-cus, has been examined by means of combined thin-layer chromatography and MALDI-TOF/MS analyses. The detailed investigation of lipid profiles has confirmed the presence of i) ether lipid phosphatidylglycerol and phosphatidylglycerophosphate methyl ester as main lipid components, ii) both C 20 and C 25 isopranoid chains in the lipid core and yielded new findings on membrane lipids of these unusual organisms. Besides some novel minor or trace phospholipids and glycolipids, data indicate the presence of ether lipid cardiolipin variants constituted by different combinations of C 20 and C 25 isopranoid chains, never before described in ar-chaea. The role of C 25 isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.

This article is available online at http://www.jlr.org erophospholipids, allowing a rapid, sensitive, and highly informative approach for the direct analysis of cell lipid extracts ( 2 ). Notably the use of the novel matrix 9-aminoacridine in MALDI-TOF/MS enables the fast and reliable detection of anionic phospholipids and also cardiolipin species ( 2 ).

The importance of the content of anionic phospholipids (cardiolipin, CL and phosphatidylglycerol, PG) in the response of B. subtilis to different stress conditions, such as high salinity, anaerobic conditions, inhibition of respiratory chain and lack of nutrients, has been examined. Total lipids have been extracted from control and stressed BS168 cells and analysed by combining TLC and MALDI-TOF mass spectrometry.