Hydrophilic interaction and reversed phase mixed-mode liquid chromatography coupled to high resolution tandem mass spectrometry for polar lipids analysis

A hydrophilic interaction liquid chromatography (HILIC) fused-core column (150 × 2.1 mm ID, 2.7 μm particle size) and a short reversed-phase liquid chromatography (RPLC) column (20 mm × 2.1 mm ID, 1.9 μm) were serially coupled to perform mixed-mode chromatography (MMC) on complex mixtures of phospholipids (PL). Mobile phase composition and gradient elution program were, preliminarily, optimized using a mixture of phosphatidylcholines (PC), phosphatidylethanolamines (PE), their corresponding lyso-forms (LPC and LPE), and sphingomyelins (SM). Thus a mixture of PC extracted from soybean was characterized by MMC coupled to electrospray ionization (ESI) high-resolution Fourier-transform mass spectrometry (FTMS) using an orbital trap analyzer. Several previously undiscovered PC, including positional isomers (i.e. 16:0/19:1 and 19:1/16:0) of PC 35:1 and skeletal isomers (i.e. 18:1/18:2 and 18:0/18:3) of PC 36:3 were identified. Therefore, high-resolution MS/MS spectra unveiled the occurrence of isomers for several overall side chain compositions. The proposed MMC-ESI-FTMS/MS approach revealed an unprecedented capability in disclosing complexity of an actual lipid extract, thus representing a very promising approach to lipidomics

Electrosynthesis of conducting poly(o-aminophenol) films on Pt substrates: a combined electrochemical and XPS investigation

Conducting poly(o-aminophenol) films (PoAP) have been electrosynthesized onto Pt substrates by cyclic voltammetry from oAP in acidic solution (HClO4 0.1 M/KClO4 0.1 M, pH 1.1). The electrosynthesis mechanism underlying the electrochemical oxidation of oAP has been investigated by in situ (Cyclic Voltammetry (CV) and Electrochemical Quartz Crystal Microbalance (EQCM)) and ex situ (X ray Photoelectron Spectroscopy (XPS)) techniques. In depth studies were performed through the characterization of polymeric layers grown up to 5, 8 and 125 voltammetric cycles. Following the initial formation of radical cations and their coupling, an induction period was noticed during which no net mass deposition occurs but rather a chemical rearrangement of adsorbed linear dimers to give aminophenoxazine like closed structures. Our experimental findings evidenced the necessity to completely reduce the linear dimer in order to allow its cyclization which is preliminary to polymer formation. We assumed that aminophenoxazine units initiate polymerization and then the addition of oxidized monomers gradually becomes the predominant route for polymer chains propagation. After 125 cycles a thickness of about 54-62 nm was estimated by EQCM. XPS investigation supported the presence of linear dimers during the induction period and allowed to attribute unambiguously a phenazine-like closed structure to the finite film. The reduced state of PoAP was characterized and showed nitrogen functionalities with binding energies intermediate between fully protonated and neutral ones. The presence of carbonyls terminal groups was also stated together with hydrating water. Heating experiments in ultra-high vacuum (UHV) conditions have revealed water molecules so strongly entrapped along the polymer chain through hydrogen bonds to be removed only at 200 °C

Ancient pottery from archaeological sites in southern Italy: First evidence of red grape product markers

The chemical analysis of tartaric acid (TA) and syringic acid (SA), as grape product markers in ancient ceramic vessels from the sites of Manduria and Torre di Satriano (southern Italy), was successfully performed. Firstly, the fragmentation behaviour of TA and SA as deprotonated molecules, [M-H]-, obtained by collision-induced dissociation, was investigated. Then, reversed-phase liquid chromatog-raphy (RPLC) with electrospray ionization (ESI) in negative ion mode, using a quadrupole linear ion trap in multiple reaction monitoring (MRM), was employed. A binary mobile phase composed of water-acetonitrile with 0.1% (v/v) acetic acid enabled the optimum ESI effciency of SA, greatly improving its identifcation when it occurs in trace amounts. Chemical analysis of ancient pottery fragments is a valid method for establishing the existence of preserved organic residues, which is valuable new evidence for the culture and customs of ancient populations, in this case those of southern Italy. The proposed RPLC-ESI-MRM method allowed a systematic investigation of ceramic fragments of both archaeological sites, thus providing positive evidence for the presence of TA and SA as grape product markers in storage vessels dating back to the ninth to third centuries BC

Unveiling the compositional variety of cardiolipins in Rhodobacter sphaeroides by liquid chromatography with electrospray ionization and multistage collision-induced dissociation mass spectrometry

Cardiolipins (CL) contained in the lipid extracts of the photosynthetic bacterium Rhodobacter sphaeroides (strain R26) were systematically characterized by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry, performed in single (MS), tandem (MS/MS) and sequential (MS3) modes using a linear ion trap mass spectrometer. The total number of carbon atoms and C=C bonds of each CL and, subsequently, those related to each of the constituting phosphatidic acid (PA) units, along with the location of the latter on the central glycerol backbone, were inferred from MS and MS/MS data, respectively. Moreover, the composition and location of both acyl chains on the glycerol backbone of each PA unit was obtained by MS3 measurements, an approach used for the first time for the structural elucidation of CL in R. sphaeroides. As a result, an unprecedented profile of CL in this bacterium was obtained, with 27 main species characterized, many of which are represented by compositional or regiochemical isomers. Interestingly, such a variability is generated from a limited number of different acyl chains, either saturated (i.e. 12:0, 16:0, 17:0, 18:0, 19:0) or mono-unsaturated (16:1, 18:1). The absence of polyunsaturated chains, more susceptible to oxidation damage, appeared to be indirectly related to the lack of carotenoids potentially acting as antioxidant agents, a specific feature of R. sphaeroides R26. The occurrence of odd-numbered acyl chains was ascribed to the need to guarantee membrane fluidity, through a less compact packing of CL, thus compensating for the lack of CL bearing polyunsaturated side chains

Untargeted lipidomics for evaluating fish authenticity: the case of wild-caught and farmed species of Sparus aurata

Aim: This study aims to develop sensitive and reliable analytical technologies to enable the distinction between wild-caught and farmed fish through appropriate molecular markers to protect consumers from fraudulent fish labelling. Gilthead sea bream, Sparus aurata L. (S. aurata L.), is a very common fish used as foodstuff worldwide and globally produced in aquaculture in the Mediterranean basin. Wild-caught and farmed species are very different in feed and lifestyle and the quality and safety of these products strongly depend on fish growth, processing history, and storage conditions. Methods: Hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform mass spectrometry (FTMS; HILIC-ESI-FTMS) was employed to discriminate the phospholipidome profiles of fillets extracts of wild-caught from farmed gilthead sea breams. Results: The untargeted approach led to the annotation of a total of 216 phospholipids (PLs), namely 65 phosphatidylethanolamines (PEs), 27 lyso-PEs (LPEs), 61 phosphatidylcholines (PCs), 34 lyso-PCs (LPCs), and 29 sphingomyelins (SMs). Untargeted lipidomics data were investigated by principal component analysis (PCA) and K-means clustering. Lyso-PLs (LPLs) of PEs and PCs including ether-linked side chains were found as discriminating markers between the two types of fish samples. The PLs that were most responsible for distinguishing between the lipid extracts of farmed and wild S. aurata fillets were successfully characterized by tandem mass spectrometry (MS/MS). The analysis revealed that wild fillet lipid extracts contained some PE exhibiting ether bonds (PE P-), viz. 16:0, 18:0, 18:1, and 18:2 and polyunsaturated fatty acyl chains (i.e., 22:6 and 22:5). In farmed species, the estimated abundance ratios of fatty acyl chains 20:4/18:2 and 22:6/20:5 were 0.9 and 0.05, respectively. However, in wild-caught fish, these ratios were found to be two-fold higher and four-fold higher, respectively. Conclusions: This work demonstrates that the combination of HILIC-ESI-FTMS and chemometrics can serve as a valuable tool for evaluating fish authenticity and assessing quality concerns by monitoring specific lipid ratios

Synthesis and MALDI MS/MS characterization of a cis-diaminocyclohexyl Pt(II)-vitamin B12 complex potentially useful for targeted drug delivery

The water-soluble vitamin B12 is essential for most organisms despite being produced by only a few bacteria. The demand for vitamin B12 is particularly high at sites of enhanced proliferation, such as cancer cells, making it particularly attractive as targeting agent. Indeed, the use of vitamin B12 for selectively delivering radioisotopes or various cytotoxic agents into cancer cells has already been actively investigated [1-3]. To these purposes vitamin B12 needs to be derivatized by the introduction of modifiers, such as ligands or receptor binding molecules. Coordination to the ribose ring and oxidative alkylation are the most common synthetic routes. Interestingly, vitamin B12 has been used to form adducts with Tc and Re complexes linked to the axial positions of the cobalt center [1], demonstrating that its Co(III)-CN moiety tends to bridge a second metal (M) ion to form a {Co-CN- M} unit. Cisplatin (cis-diammine-dichloro-Pt(II)) is one of the most potent agents against a wide variety of solid tumors, its cytotoxicity being mediated by formation of DNA adducts [4]. Thus the possibility of using vitamin B12 as a ligand for cisplatin (and its analogues) would be highly desirable in view of a targeted delivering of anticancer drugs [2]. In this communication, we demonstrate that Co-CN in vitamin B12 bridges to Pt(II) center yielding stable complexes with the central structural feature {Co-CN-Pt}. The formation of the cis-Pt(II) complex with vitamin B12 (m/z 1663.647) was investigated by matrix assisted laser desorption/ionization mass spectrometry, MALDI MS in positive ion mode, using 4-chloro-α-cyanocinnamic acid as a matrix [5]. Structural characterization was accomplished by collision-induced dissociation (ToF/ToF) MS analysis

Effect of Storage and Extraction Protocols on the Lipid and Fatty Acid Profiles of Dicentrarchus labrax Brain

Fish brain might represent an interesting raw material since it is particularly enriched in lipids (e.g., phosphatidylcholines (PCs)) that are valuable food additives and nutraceuticals. A chloroform-free solvent extraction protocol based on 2-methoxy-2-methylpropane also known as methyl tert-butyl ether (MTBE) was evaluated and compared with CHCl3:MeOH mixtures of the classical Bligh-Dyer method. Phospholipids (PLs) and sphingolipids (SPs) occurring in the brain extracts of farmed European sea bass fish (Dicentrarchus labrax) were identified/analyzed by flow injection-electrospray ionization-mass spectrometry (FI-ESI-MS), whereas the content and identity of fatty acids (FA) were obtained by gas chromatography-MS upon derivatization as FA methyl esters. On the basis of these preliminary results, the total contents of PL and SP, as well as their relative abundances, were not significantly affected by the extraction protocol used. The most abundant lipid class was the PC with a mean ± SD content (expressed as nmol/mg protein) of 507 ± 89, followed by phosphoethanolamines (195 ± 41), ceramides (35 ± 9), sphingomyelins (9 ± 4), lyso-phosphatidylcholines (8 ± 2), and phosphatidylglycerols (1.8 ± 0.8). In terms of pre-processing strategy of fish brain samples, two preservation methods, i.e., freezing and freeze-drying, were also compared. Lyophilization led to a partial degradation of PC to their corresponding lyso-form, most likely as a result of endogenous phospholipase activity

Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MSn)

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the α-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MSn, n = 2-4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H]- at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H]- ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides

Deletion of gene coding for mitochondrial citrate carrier in C. elegans impairs the acetylcholine synthesis

The mitochondrial citrate carrier (CIC) encoded by the SLC25A1 gene, catalyzes the export of citrate from mitochondria to the cytosol where it is broken into acetyl-CoA and oxaloacetate. Acetyl-CoA can be used as building block for de novo synthesis of fatty acids or, in motoneurons, can be condensed to choline to give acetylcholine. Pathogenic SLC25A1 variants were identified in several patients (1, 2) that suffered from a severe neurodevelopmental syndrome (1) and CIC deficiency has been classified as an inborn disorder of metabolism (OMIM: 615182) whose hallmark is combined D-2- and L-2-hydroxyglutaric aciduria (2). More recently we reported a novel homozygous mutation in the SLC25A1 gene in an affected sib pair. Both patients presented with myasthenia and impaired neuromuscular junction (NMJ) transmission whilst no neurodevelopment disorder has been observed (3). Interestingly, the identified mutation in sib pair caused a milder activity impairment than the previously reported mutations suggesting a fundamental role of CIC in neuromuscular transmission whose defect was previously masked by the harsher phenotypes. Signal transmission at Neuromuscular junction (NMJ) relies on massive synthesis in motor neurons of acetylcholine. While the choline can be recycled after neurotransmitter release and breakdown in the synaptic cleft, the acetylCoA must be continuously generated by oxidative metabolism in mitochondria. Knocking down the SLC25A1 orthologues by injection of antisense morpholino oligonucleotides in zebrafish embryos showed short and erratic outgrowth of motor axons toward muscle fiber at NMJ suggesting that transmission impairment could be related to pre-synaptic nerve terminal abnormalities. Using the CRISPR/CAS9 approach we obtained stable lines of Caenorhabditis elegans knocked-out in the SLC25A1 ortholog that showed resistance to levamisole, a nicotinic acetylcholine receptor agonist, that causes continued stimulation of the worm muscles, leading to paralysis. This phenotype was, at least in part, rescued by the expression of wild-type SLC25A1 under the control of a neuron-specific promoter, strongly pointing towards an underlying pre-synaptic defect. qPCR analysis showed an up-regulation of genes involved in presynaptic acetylcholine biosynthesis and its release in the synaptic cleft, and a down-regulation of acetylcholine esterase. Furthermore, the amount of acetylcholine is decreased by about 50% in KO strain. Lipidomic analysis by mass spectrometry (HILIC-ESI-FTMS) showed no gross defect in lipogenesis. Altogether these data demonstrate a conserved role of CIC in neuromuscular transmission. Furthermore, our results validate the worm C. elegans as an animal model suitable for further study of the molecular and cellular underpinnings of the NMJ transmission defect associated to CIC deficiency 1. Edvardson et al. 2013 J Med Genet 50:240-5 2. Nota et al. 2013 Am J Hum Genet 92:627-31 3. Chaouch et al. 2014 J Neuromuscul Dis 1:75-9