Roberto Angelini

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.

Novel ether lipid cardiolipins in archaeal membranes of extreme haloalkaliphiles

The lipidome of two extremely haloalkaliphilic archaea, Natronococcus occultus and Natronococcus amylolyticus, 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 archaea. The role of C(25) isopranoid chains in the adaptation to high pH gradients in the presence of very high salt concentrations is discussed.

MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine

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.

Lipidomic Analysis of Porcine Olfactory Epithelial Membranes and Cilia

The use of the matrix 9-aminoacridine has been recently introduced in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of both anionic and cationic phospholipids. In the present study, we take advantage of this technique to analyze the lipids of porcine olfactory mucosa and a membrane fraction enriched in cilia. Thin-layer chromatography (TLC) and 31P-NMR analyses of the lipid extracts were also performed in parallel. MALDI-TOF-MS allowed the identification of lipid classes in the total lipid extract and individual lipids present in the main TLC bands. The comparison between the composition of the two lipid extracts showed that: (1) cardiolipin, present in small amount in the whole olfactory mucosa lipid extract, was absent in the extract of membranes enriched in olfactory cilia, (2) phosphatidylethanolamine species were less abundant in ciliary than in whole epithelial membranes, (3) sulfoglycosphingolipids were detected in the lipid extract of ciliary membranes, but not in that of epithelial membranes. Our results indicate that the lipid pattern of ciliary membranes is different from that of whole-tissue membranes and suggest that olfactory receptors require a specific lipid environment for their functioning.

Coupled TLC and MALDI-TOF/MS analyses of the lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus.

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.

Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome

Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of cardiolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a “CL fingerprint” and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells.

Adjusting membrane lipids under salt stress: the case of the moderate halophilic organism Halobacillus halophilus

The lipid composition of Halobacillus halophilus was investigated by combined thin-layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses of the total lipid extract. Main polar lipids were found to be sulfoquinovosyldiacylglycerol and phosphatidylglycerol, while cardiolipin was a minor lipid together with phosphatidic acid, alanyl-phosphatidylglycerol and two not yet fully identified lipid components. In addition the analyses of residual lipids, associated with denatured proteins after the lipid extraction, revealed the presence of significant amounts of cardiolipin, indicating that it is a not readily extractable phospholipid. Post decay source mass spectrometry analyses allowed the determination of acyl chains of main lipid components. On increasing the culture medium salinity, an increase in the shorter chains and the presence of chain unsaturations were observed. These changes in the lipid core structures might compensate for the increase in packing and rigidity of phospholipid and sulfoglycolipid polar heads in high-salt medium, therefore contributing to the homeostasis of membrane fluidity and permeability in salt stress conditions.

Relationship between cardiolipin metabolism and oxygen availability in Bacillus subtilis

We report changes of the content of anionic phospholipids in Bacillus subtilis in response to hypoxic conditions and inhibition of terminal respiration. Cardiolipin accumulates rapidly when bacteria are suspended in non‐growth medium under reduced aeration or exposed to the inhibitor cyanide; the increase of cardiolipin occurs at the expense of its precursor phosphatidylglycerol and is temperature‐dependent. Depending on the extent of hypoxic stress, membranes containing different levels of cardiolipin can be isolated from B. subtilis cells. The NADH oxidase activity in cardiolipin‐enriched membranes is cyanide‐resistant; furthermore O2 consumption measurements indicated that cardiolipin‐enriched cells are resistant to cyanide. Results point out a possible interdependence between the effect of cyanide on cardiolipin metabolism and the effect of cardiolipin on the effectiveness of cyanide inhibition.

Isolation of Squarebop I bacteriorhodopsin from biomass of coastal salterns

Squarebop I bacteriorhodopsin is a light-activated proton pump present in the membranes of the archeon Haloquadratum walsbyi, a square-shaped organism representing 50-60% of microbial population in the crystallizer ponds of the coastal salterns. Here we describe: (1) the operating mode of a bioreactor designed to concentrate the saltern biomass through a microfiltration process based on polyethersulfone hollow fibers; (2) the isolation of Squarebop I bacteriorhodopsin from solubilized biomass by means of a single chromatographic step; (3) tightly bound lipids to the isolated and purified protein as revealed by MALDI-TOF/MS analysis; (4) the photoactivity of Squarebop I bacteriorhodopsin isolated from environmental samples by flash spectroscopy. Yield of the isolation process is 150 μg of Squarebop I bacteriorhodopsin from 1l of 25-fold concentrated biomass. The possibility of using the concentrated biomass of salterns, as renewable resource for the isolation of functional bacteriorhodopsin and possibly other valuable bioproducts, is briefly discussed.

Impact of Lipidomics on the Microbial World of Hypersaline Environments

Mass spectrometry analysis of the lipid extracts of saltern biomass represents a powerful tool to quickly obtain information on the presence of various archaeal and bacterial microorganisms in saltern ponds. In the last years, ESI-MS lipid profiling by a shotgun lipidomic approach has allowed the discovery of new lipid molecules in the membranes of archaeal and bacterial halophilic microorganisms. The recent introduction of the MALDI-TOF/MS technique in lipid analysis offers further possibilities of implementing the knowledge of lipid biology of halophilic prokaryotes. In this chapter, the structures and functional role of new lipids of extreme halophilic Archaea and Bacteria are illustrated in relation to adaptation to osmotic stress.