fingerprinting-cardiolipin-leukocytes-mass-spectrometry-for-rapid

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial ...

University of Bari Aldo Moro

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.

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

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed.

The light-activated proton pump Bop I of the archaeon Haloquadratum walsbyi.

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.

Isolation of Squarebop I bacteriorhodopsin from biomass of coastal salterns.

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at T(m) = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I-mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10(-8) J/m(2). In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.

Morphology, biophysical properties and protein-mediated fusion of archaeosomes.

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.

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

The structures of archaeal glycerophospholipids and glycolipids are unique in that they consist of phytanyl substituents ether linked to the glycerol backbone, imparting stability to the molecules. In this contribution, we described multiple-stage linear ion-trap combined with high resolution mass spectrometry toward structural characterization of this lipid family desorbed as lithiated adduct ions or as the [M-H](-) and [M-2H](2-) ions by ESI. MS(n) on various forms of the lithiated adduct ions yielded rich structurally informative ions leading to complete structure identification of this lipid family, including the location of the methyl branches of the phytanyl chain. By contrast, structural information deriving from MS(n) on the [M-H](-) and [M-2H](2-) ions is not complete. The fragmentation pathways in an ion-trap, including unusual internal loss of glycerol moiety and internal loss of hexose found for this lipid family were proposed. This mass spectrometric approach provides a simple tool to facilitate confident characterization of this unique lipid family.

Structural studies on archaeal phytanyl-ether lipids isolated from membranes of extreme halophiles by linear ion-trap multiple-stage tandem mass spectrometry with electrospray ionization.

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.

Relationship between cardiolipin metabolism and oxygen availability in Bacillus subtilis.

Polar membrane lipids of an archaeal microorganism recently isolated from the natural salt lake Fuente de Piedra (Málaga, Spain) have been studied by means of TLC in combination with MALDI-TOF mass spectrometry. The major phospholipids are the ether lipids phosphatidylglycerophosphate methyl ester and phosphatidylglycerosulfate, while phosphatidylglycerol is barely detectable; in addition the bisphosphatidylglycerol (archaeal cardiolipin) has been detected for the first time in a representative of the genus Halobellus. The structures of glycolipids, including a glycosyl-cardiolipin, have been elucidated by post source decay (PSD) mass spectrometry analysis. Besides the monosulfated diglycosyl diphytanylglyceroldiether, two variants of a bis-sulfated diglycosyl diphytanylglyceroldiether have been identified; furthermore the glycosyl-cardiolipin is found to have the same structure of the analogue present in Halorubrum trapanicum and Haloferax volcanii. The role of the abundant sulfated glycolipids in facing high extracellular salinity is discussed. 

Deciphering archaeal glycolipids of an extremely halophilic archaeon of the genus Halobellus by MALDI-TOF/MS.

The aim of this study was to explore the possibility of using an archaeal microorganism as a host system for expressing mammalian olfactory receptors (ORs). We have selected the archaeon Haloferax volcanii as a cell host system and one of the most extensively investigated OR, namely I7-OR, whose preferred ligands are short-chain aldehydes, such as octanal, heptanal, nonanal. A novel plasmid has been constructed to express the rat I7-OR, fused with a hexahistidine-tag for protein immunodetection. The presence of the recombinant receptor at a membrane level was demonstrated by immunoblot of the membranes isolated from the transgenic archaeal strain. In addition, the lipid composition of archaeonanosomes containing ORs has been characterized in detail by High-Performance Thin-Layer Chromatography (HPTLC) in combination with Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight/Mass Spectrometry (MALDI-TOF/MS) analysis. 

Haloferax volcanii, as a Novel Tool for Producing Mammalian Olfactory Receptors Embedded in Archaeal Lipid Bilayer.

Barth syndrome is a rare X-linked disease affecting less than 200 individuals worldwide. Several comorbidities have been associated with the pathology and, among those, cardiac myopathy and neutropenia are the most life threatening. The appropriate nutritive support is important to sustain the everyday life of Barth syndrome patients given the chronic fatigue they experience. Since they often prefer salty and fried food, and avoid vegetables and fruits, their eating habit and food preferences do not always provide the proper amount of vitamins and amino acids. It has been indeed reported that Barth syndrome patients have altered taste sensitivity. As olfaction also contributes to food consumption and flavor perception, we decided to investigate their olfactory abilities using the "Sniffin' sticks' extended test". We found no significant difference in any of the tested olfactory abilities between the group of Barth syndrome patients and the healthy controls. In summary, altered food preference of Barth boys could not be easily explained with an altered olfactory perception.

Assessing olfactory functions in patients with Barth syndrome.

The brine shrimp  is an interesting experimental system for studies of developmental processes. Hatching of dormant cysts gives rise to shrimp larvae called nauplii, characterized by numerous naupliar stages representing the first forms of brine shrimp life cycle. Here combined Thin Layer Chromatography (TLC) and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) analyses have been performed to gain information on the lipid profiles of cysts and two naupliar stages. Lipid bands isolated after preparative TLC of the lipid extracts have been analyzed to detect various species of each lipid class; in addition PostSource Decay (PSD) analyses allowed the identification of phospholipid chains. We compared the relative abundance of various polar and neutral lipid species in the lipid extracts, proving for the first time that during the development of nauplii there is an increase of cardiolipin (CL) and lysophospholipid levels; in parallel, the amount of phosphatidylcholine (PC) decreases. In addition, as regards neutral lipids, we found an increase of diacylglycerols (DAGs) in correspondence of the decrease of triacylglycerols (TAGs). Data reflect the fact that naupliar stages, being an active form of life, are more metabolically active and offer a platform to develop further studies on the importance of lipid metabolic pathways and bioactive lipids during the development.

Lipid Profile Changes During the Development of , From Cysts to the First Two Naupliar Stages.

The reduction of sperm motility and count, or oligoasthenozoospermia, is one of the major causes of reduced fertility or infertility in men. Lipid composition of spermatozoa is important in determining their functional characteristics, in particular on motility, acrosomal exocytosis or fusogenic properties of the sperm. Here we investigated the levels of semen lipids in 11 infertile patients with severe oligoasthenozoospermia and 9 normozoospermic subjects with normal motility values. Sperm polar and neutral lipids were analyzed by thin-layer chromatography (TLC) and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Semen of patients with oligoasthenozoospermia showed a reduction of the degree of fatty acid unsaturation in the phospholipids chains that might affect the membrane fluidity. Furthermore, a significant higher cholesterol sulfate/seminolipid ratio was found in semen of oligoasthenozoospermic patients than in subjects with normal motility values, suggesting a critical role of sulfolipids in semen quality. The results may facilitate the understanding of the role of lipids on male fertility and offer interesting perspectives to find innovative treatments for oligoasthenozoospermia.

Alteration of Cholesterol Sulfate/Seminolipid Ratio in Semen Lipid Profile of Men With Oligoasthenozoospermia.

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable  propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

Simona Lobasso

Department of Basic Medical Sciences,
Neuroscience and Sense Organs,
Department of Basic Medical Sciences, Neuroscience and Sense Organs

University of Bari Aldo Moro

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Simona Lobasso

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Basic Medical Sciences,Neuroscience and Sense Organs,Department of Basic Medical Sciences, Neuroscience and Sense Organs

University of Bari Aldo Moro

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Department of Basic Medical Sciences,
Neuroscience and Sense Organs,
Department of Basic Medical Sciences, Neuroscience and Sense Organs