MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

Di‐ and tri‐phenyltin chlorides transfer across a model lipid bilayer

A compound's ability to penetrate the plasma membrane of a cell is the critical parameter that determines its potential to become a biologically potent factor. A well‐known group of organotin compounds that exhibit toxic properties in relation to biological systems are phenyltins. There are as yet no studies that in a direct manner have established whether organotin compounds such as diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT) diffuse, or not, through the lipid bilayer, although we know that at least some organotins absorb in both liposome and biological membranes. In this paper we present a series of experiments that show transfer of these compounds across the lipid membrane using the stopped‐flow technique. The results obtained demonstrate that DPhT and TPhT first adsorb onto the lipid bilayer surface, in a diffusion‐controlled manner and within a very short time (0.05 s), whereas the membrane crossing was observed to be on the order of a minute. The adsorption process was easily fitted with a single exponential for both the compounds studied, indicating a single process phenomenon. The longer time kinetics (characteristic of membrane crossing) showed a complex dependence on compound concentration and the presence of cholesterol in the membrane. On passing from the outer to the inner surface of the bilayer, organotins undergo desorption and enter the liposome interior, which has been shown in lipid monolayer desorption studies. In conclusion, it can be stated that amphiphilic DPhT and TPhT permeate the liposome membrane. Copyright © 2005 John Wiley & Sons, Ltd.     

Substrate‐induced formation of a recognition structure in a four‐polypeptide–lipid monolayer system

Poly(γ‐methyl L ‐glutamate)s with Ser, His, Asp, and Glu residues at the amino terminal as the serine protease catalytic site were prepared. The number‐average degree of polymerization of the polypeptides was 51. A dipalmitoylphosphatidylcholine monolayer containing the polypeptides was formed at the air–water interface and was transferred onto gold‐deposited glass plates. The binding of N‐acetyltyrosine ethyl ester, a typical substrate of the serine protease, to the monolayer was characterized by surface plasmon resonance measurements. The four‐polypeptide–lipid monolayer system conditioned on an aqueous solution containing the substrate N‐acetyltyrosine ethyl ester exhibited Langmuir‐type binding of the substrate. Its binding constant of 6.1 × 10⁴ M⁻¹ was about 20 times larger than that observed for a monolayer prepared on pure water. The behavior may have arisen from a substrate‐induced rearrangement of the four kinds of polypeptides in the monolayer, forming a substrate‐binding structure similar to that found in serine protease. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2186–2191, 2000     

Ultrasound‐enhanced and microwave‐assisted extraction of lipid from Dunaliella tertiolecta and fatty acid profile analysis

Microalgal lipid is considered as a potential biodiesel resource due to its advantages compared to other bioresources. The production of biofuel from microalgae includes several stages like microalgae cultivation, biomass harvest, biomass treatment, lipid extraction, and the ultimate biodiesel synthesis. Lipid extraction is closely associated with the productivity and cost of energy production. In the present study, lipid of green algae Dunaliella tertiolecta was extracted by chemical agents with involvement of ultrasound and microwave. The optimization of experimental conditions was carried out by response surface methodology and orthogonal test design. Using the ultrasonic technique, an extraction rate of 45.94% was obtained under the optimum conditions of ultrasonic power 370 W, extraction time 5 min and liquid/solid ratio 125 mL/g. The extraction rate of 57.02% was obtained by the means of microwave assistance under the optimized conditions of extraction time 160 s, microwave power 490 W and liquid/solid ratio 100 mL/g. The comparison of the two results indicated microwave was more effective than ultrasound in extracting process. When the two techniques were utilized in combination, the optimized condition was ultrasonic power 320 W, ultrasonic time 4 min, microwave power 280 W, microwave time 120 s and liquid/solid ratio 100 mL/g, and the extraction rate was 49.97%.     

HPLC‐FLD determination of NBD‐cholesterol,its ester and other metabolites in cellular lipid extracts

22‐[N(?7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐23,24‐bisnor‐5‐cholen‐3β‐ol (NBD‐cholesterol), a fluorescent cholesterol analog, was an extragenous cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high‐performance liquid chromatography/fluorescence detection (HPLC‐FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625–10.0 µg/mL (r² = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD‐cholesteryl oleate, two additional cellular metabolites of NBD‐cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco‐2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl‐CoA:cholesterol acyltransferase‐1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC‐C8‐434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco‐2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC‐FLD method is a powerful tool for simultaneous quantification of intracellular NBD‐cholesterol and its oleoyl‐ester. Copyright © 2013 John Wiley & Sons, Ltd.     

13C‐TmDOTA as versatile thermometer compound for solid‐state NMR of hydrated lipid bilayer membranes

Recent advances in solid‐state nuclear magnetic resonance (NMR) techniques, such as magic angle spinning and high‐power decoupling, have dramatically increased the sensitivity and resolution of NMR. However, these NMR techniques generate extra heat, causing a temperature difference between the sample in the rotor and the variable temperature gas. This extra heating is a particularly crucial problem for hydrated lipid membrane samples. Thus, to develop an NMR thermometer that is suitable for hydrated lipid samples, thulium‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate (TmDOTA) was synthesized and labeled with ¹³C (i.e., ¹³C‐TmDOTA) to increase the NMR sensitivity. The complex was mixed with a hydrated lipid membrane, and the system was subjected to solid‐state NMR and differential scanning calorimetric analyses. The physical properties of the lipid bilayer and the quality of the NMR spectra of the membrane were negligibly affected by the presence of ¹³C‐TmDOTA, and the ¹³C chemical shift of the complex exhibited a large‐temperature dependence. The results demonstrated that ¹³C‐TmDOTA could be successfully used as a thermometer to accurately monitor temperature changes induced by ¹H decoupling pulses and/or by magic angle spinning and the temperature distribution of the sample inside the rotor. Thus, ¹³C‐TmDOTA was shown to be a versatile thermometer for hydrated lipid assemblies. Copyright © 2015 John Wiley & Sons, Ltd.     

Centerband‐only analysis of rotor‐unsynchronized spin echo for measurement of lipid 31P chemical shift anisotropy

Structural diversity and molecular flexibility of phospholipids are essential for biological membranes to play key roles in numerous cellular processes. Uncovering the behavior of individual lipids in membrane dynamics is crucial for understanding the molecular mechanisms underlying biological functions of cell membranes. In this paper, we introduce a simple method to investigate dynamics of lipid molecules in multi‐component systems by measuring the ³¹P chemical shift anisotropy (CSA) under magic angle spinning (MAS) conditions. For achieving both signal separation and CSA determination, we utilized a centerband‐only analysis of rotor‐unsynchronized spin echo (COARSE). This analysis is based on the curve fitting of periodic modulation of centerband intensity along the interpulse delay time in rotor‐unsynchronized spin‐echo experiments. The utility of COARSE was examined by using phospholipid vesicles, a three‐component lipid raft model system, and archaeal purple membranes. We found that the apparent advantages of this method are high resolution and high sensitivity given by the moderate MAS speed and the one‐dimensional acquisition with short spin‐echo delays. COARSE provides an alternative method for CSA measurement that is effective in the investigation of lipid polymorphologies. Copyright © 2015 John Wiley & Sons, Ltd.     

A rapid one‐step method for the characterization of membrane lipid remodeling in Francisella using matrix‐assisted laser desorption ionization time‐of‐flight tandem mass spectrometry

Lipids are essential components of all bacterial membranes. The most common membrane‐associated lipids in Gram‐negative bacteria are phospholipids and lipid A, the hydrophobic anchor of lipopolysaccharide. Diversity in these lipids arises through structural modifications that include changes in the length and location of fatty acids, and the addition of phosphate and carbohydrate moieties. Analysis of individual structural modifications normally requires large quantities of starting material and multiple methods for the isolation, hydrolysis, and analysis. In this study, we developed a novel one‐step protocol for the combined isolation of phospholipids and lipid A from Francisella subspecies followed by analysis using matrix‐assisted laser desorption ionization time‐of‐flight tandem mass spectrometry. The total time for lipid isolation and analysis was approximately 15 min and with a lower limit of detection of approximately 100 ng of purified lipid. This protocol identified the major lipid structures using both wild‐type Ft subspecies strains and lipid A biosynthesis mutants. We also determined the relative levels of individual lipid A and phospholipids after growth under conditions that mimic the mammalian infection process. This analysis showed that the bacterial membranes remodeled rapidly to adapt to changes in environmental growth conditions and may be important for Francisella pathogenesis. Copyright © 2011 John Wiley & Sons, Ltd.     

A sensitive liquid chromatography/mass spectrometry‐based assay for quantitation of amino‐containing moieties in lipid A

A novel sensitive liquid chromatography/mass spectrometry‐based assay was developed for the quantitation of aminosugars, including 2‐amino‐2‐deoxyglucose (glucosamine, GlcN), 2‐amino‐2‐deoxygalactose (galactosamine, GalN), and 4‐amino‐4‐deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino‐containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed‐phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10‐fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium‐limited versus magnesium‐replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium‐replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino‐containing moieties in lipid A from a variety of bacterial sources. Copyright © 2009 John Wiley & Sons, Ltd.     

Loading characteristics and chemical stability of headgroup‐functionalized poly(ethylene glycol)‐lipid ligand tethers on polypropylene capillary‐channeled polymer fibers

Polypropylene capillary‐channeled polymer fibers have been modified by adsorption of headgroup‐functionalized poly(ethylene glycol)‐lipids to generate a species‐specific stationary phase. In order to study ligand binding characteristics, a fluorescein‐labeled poly(ethylene glycol)‐lipid was used as a model system. Breakthrough curves and frontal analysis were employed to characterize the surface loading characteristics across a range of lipid concentrations and mobile phase flow rates. Efficient mass transfer and fluid transport yield a linear adsorption isotherm up to the maximum loading concentration of 3 mg/mL, at a linear velocity of 57.1 mm/s. Under these conditions, the dynamic binding capacity was found to be 1.52 mg/g of fiber support. Variation of the linear velocity from 8.6 to 57.1 mm/s showed only small changes in breakthrough volume. The maximum capacity of 1.8 mg/g is found under conditions of a load velocity of 34.2 mm/s and a concentration of 3 mg/mL lipid. Exposure of the lipid modified fibers to several challenge solvents reveals a chemically robust system, with only 50% acetonitrile and hexanes able to disrupt the lipid adsorption. The straightforward capillary‐channeled polymer fiber surface modification with headgroup‐functionalized lipids provides both a diverse yet practically robust ligand tethering system.     

On‐chip generation of monodisperse giant unilamellar lipid vesicles containing quantum dots

Monodispersed lipid vesicles have been used as a drug delivery vehicle and a biochemical reactor. To generate monodispersed lipid vesicles in the nano‐ to micrometer size range, an extrusion step should be included in conventional hand‐shaking method of lipid vesicle synthesis. In addition, lipid vesicles as a drug carrier still need to be improved to effectively encapsulate concentrated biomolecules such as cells, proteins, and target drugs. To overcome these limitations, this paper reports a new microfluidic platform for continuous synthesis of small‐sized (～10 μm) giant unilamellar vesicles (GUVs) containing quantum dots (QDs) as a nanosized model drug. To generate GUVs, we introduced an additional cross‐flow to break vesicles into small size. 1,2 ‐ dimyristoyl‐sn‐glycero ‐ 3 ‐ phosphocholine (DMPC) in an octanol–chloroform mixture was used in the construction of self‐assembled membrane. Consequently, we have successfully demonstrated the fabrication of monodispersed GUVs with 7?12 μm diameter containing QDs. The proposed synthesis method of cell‐sized GUVs would be highly desirable for applications such as multipurpose drug encapsulation and delivery.     

Characterization of complex,heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non‐phosphorylated lipid A by negative‐ion mode tandem mass spectrometry

Non‐phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non‐phosphorylated lipid A species under low‐energy collision‐induced dissociation conditions of an electrospray ionization quadrupole time‐of‐flight instrument. Charge‐promoted consecutive and competitive eliminations of the acyl chains and cross‐ring cleavages of the sugar residues were observed. The A‐type fragment ion series and the complementary X‐type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non‐reducing and the reducing ends, respectively, of the non‐phosphorylated lipid A backbone. Reversed‐phase liquid chromatography in combination with negative‐ion electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non‐phosphorylated lipid A. As a standard, the hexa‐acylated ion at m/z 1636 with the Escherichia coli‐type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non‐phosphorylated hexa‐ and several hepta‐, penta‐, and tetra‐acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

O‐Benzyl‐N‐tert‐butoxy­carbonyl‐N‐methyl‐l‐tyrosine

The crystal structure of the title compound, alternatively called 3‐[4‐(benzyl­oxy)­phenyl]‐2‐(N‐tert‐butoxy­car­bonyl‐N‐methyl­amino)­propi­onic acid, C₂₂H₂₇NO₅, has been studied in order to ex­amine the role of N‐methyl­ation as a determinant of peptide conformation. The conformation of the tert‐butoxy­carbonyl group is trans–trans. The side chain has a folded conformation and the two phenyl rings are effectively perpendicular to one another. The carboxyl­ate hydroxyl group and the urethane carbonyl group form a strong intermolecular O—H?O hydrogen bond.     

rac‐5‐Diphenylacetyl‐2,2,4‐trimethyl‐2,3,4,5‐tetrahydro‐1,5‐benzothiazepine and rac‐5‐formyl‐2,2,4‐trimethyl‐2,3,4,5‐tetrahydro‐1,5‐benzothiazepine

rac‐5‐Diphenylacetyl‐2,2,4‐trimethyl‐2,3,4,5‐tetrahydro‐1,5‐benzothiazepine, C₂₆H₂₇NOS, (I), and rac‐5‐formyl‐2,2,4‐trimethyl‐2,3,4,5‐tetrahydro‐1,5‐benzothiazepine, C₁₃H₁₇NOS, (II), are both characterized by a planar configuration around the heterocyclic N atom. In contrast with the chair conformation of the parent benzothiazepine, which has no substituents at the heterocyclic N atom, the seven‐membered ring adopts a boat conformation in (I) and a conformation intermediate between boat and twist‐boat in (II). The molecules lack a symmetry plane, indicating distortions from the perfect boat or twist‐boat conformations. The supramolecular architectures are significantly different, depending in (I) on C—H...O interactions and intermolecular S...S contacts, and in (II) on a single aromatic π–π stacking interaction.     

5‐Acetyl‐2‐amino‐6‐methyl‐4‐phenyl‐4H‐pyran‐3‐carbonitrile and 2‐amino‐5‐benzoyl‐6‐methyl‐4‐phenyl‐4H‐pyran‐3‐carbonitrile acetonitrile solvate

The syntheses, X‐ray structural investigations and calculations of the conformational preferences of the carbonyl substituent with respect to the pyran ring have been carried out for the two title compounds, viz. C₁₅H₁₄N₂O₂, (II), and C₂₀H₁₆N₂O₂·C₂H₃N, (III), respectively. In both mol­ecules, the heterocyclic ring adopts a flattened boat conformation. In (II), the carbonyl group and a double bond of the heterocyclic ring are syn, but in (III) they are anti. The carbonyl group forms a short contact with a methyl group H atom in (II). The dihedral angles between the pseudo‐axial phenyl substituent and the flat part of the pyran ring are 92.7 (1) and 93.2 (1)° in (II) and (III), respectively. In the crystal structure of (II), inter­molecular N—H⋯N and N—H⋯O hydrogen bonds link the mol­ecules into a sheet along the (103) plane, while in (III), they link the mol­ecules into ribbons along the a axis.