Mass spectrometry characterisation of fatty acids from metabolically engineered soybean seeds

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and down-regulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.

Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine

We describe the use of the ¹³C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein–peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a ¹³C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein–protein interaction studies. Figure

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Automated quantitative analysis of lipid accumulation and hydrolysis in living macrophages with label-free imaging

The accumulation of lipids in macrophages is a key factor that promotes the formation of atherosclerotic lesions. Several methods such as biochemical assays and neutral lipid staining have been used for the detection of lipids in cells. However, a method for real-time quantitative assessment of the lipid content in living macrophages has yet to be shown, particularly for its kinetic process with drugs, due to the lack of suitable tools for non-invasive chemical detection. Here we demonstrate label-free real-time monitoring of lipid droplets (LDs) in living macrophages by using coherent anti-Stokes Raman scattering (CARS) microscopy. In addition, we have established an automated image analysis method based on maximum entropy thresholding (MET) to quantify the cellular lipid content. The result of CARS image analysis shows a good correlation (R ²?>?0.9) with the measurement of biochemical assay. Using this method, we monitored the processes of lipid accumulation and hydrolysis in macrophages. We further characterized the effect of a lipid hydrolysis inhibitor (diethylumbelliferyl phosphate, DEUP) and determined the kinetic parameters such as the inhibition constant, K _i. Our work demonstrates that the automated quantitative analysis method is useful for the studies of cellular lipid metabolism and has potential for preclinical high-throughput screening of therapeutic agents related to atherosclerosis and lipid-associated disorders.

Evaluation of a commercial enzymatic test kit regarding the quantitative analysis of different free fatty acids

The quantitative determination of the total free fatty acids (FFAs) is an important analytical task because FFAs exhibit important physiological effects and are also relevant in many other fields, for instance, in food research. Our aim was to investigate whether a commercially available enzymatic test kit developed for the determination of FFAs in human serum is also suitable to determine different physiological and nonphysiological FFAs and to which extent the impact on the sensitivities (i.e., the accuracy by which a given FFA can be determined) differ. It will be shown that the chain length as well as the double bond content has a significant impact on the sensitivity by which a given FFA can be determined. For instance, palmitic acid (16:0) is determined with an approximately 20 times higher sensitivity in comparison to docosahexaenoic acid (22:6n-3). All data were obtained by measuring the concentrations of the FFAs by gas chromatography, and selected FFAs were also determined in a complex matrix of human serum. It is concluded that this kit is not useful if major alterations of the FFA composition of a complex mixture are expected because the individual FFAs are not detected with the same sensitivities: the concentrations of polyunsaturated FFA determined by this kit are wrong. Figure

The used enzymatic kit detects different free fatty acids with significantly different sensitivities: the number of carbon atoms and the number of double bonds massively contribute to these differences     

Simultaneous sampling of volatile and non-volatile analytes in beer for fast fingerprinting by extractive electrospray ionization mass spectrometry

By gently bubbling nitrogen gas through beer, an effervescent beverage, both volatile and non-volatile compounds can be simultaneously sampled in the form of aerosol. This allows for fast (within seconds) fingerprinting by extractive electrospray ionization mass spectrometry (EESI-MS) in both negative and positive ion mode, without the need for any sample pre-treatment such as degassing and dilution. Trace analytes such as volatile esters (e.g., ethyl acetate and isoamyl acetate), free fatty acids (e.g., caproic acid, caprylic acid, and capric acid), semi/non-volatile organic/inorganic acids (e.g., lactic acid), and various amino acids, commonly present in beer at the low parts per million or at sub-ppm levels, were detected and identified based on tandem MS data. Furthermore, the appearance of solvent cluster ions in the mass spectra gives insight into the sampling and ionization mechanisms: aerosol droplets containing semi/non-volatile substances are thought to be generated via bubble bursting at the surface of the liquid; these neutral aerosol droplets then collide with the charged primary electrospray ionization droplets, followed by analyte extraction, desolvation, ionization, and MS detection. With principal component analysis, several beer samples were successfully differentiated. Therefore, the present study successfully extends the applicability of EESI-MS to the direct analysis of complex liquid samples with high gas content.

Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar–attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms.

Bilayer lipid membranes from falling droplets

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid–liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid–alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain–Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-Å crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Differentiation of Diastereoisomers of Protected 1,2-Diaminoalkylphosphonic Acids by EI Mass Spectrometry and Density Functional Theory

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions.

Fatty acid variability in three medicinal herbs of Panax species

Background

Fatty acid profiling has been widely used in the bacteria species identification, we hypothesized that fatty acid characteristics might discriminate the Panax herbs according to species. To test the hypothesis, fatty acids of Panax species, including Panax ginseng, Panax notoginseng and Panax quinquefolius, were characterized and compared using gas chromatography–mass spectrometry (GC-MS) followed by multivariate statistical analysis.

Results

The content of investigated 11 fatty acids, including myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, arachidic acid and eicosadienoic acid, obviously varied among three species, suggesting each species has its own fatty acid pattern. Principal component analysis and hierarchical clustering analysis according to the absolute and relative contents of fatty acids, showed that 30 tested samples could be clearly differentiated according to the species.

Conclusions

These findings demonstrated that GC-MS-based fatty acid profiling coupled with multivariate statistical analysis provides reliable platform to classify these three Panax species, which is helpful for ensuring their safety and efficacy.     

Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

Qualitative Gas Chromatography–Mass Spectrometry Analyses Using Amines as Chemical Ionization Reagent Gases

Ammonia is a very useful chemical ionization (CI) reagent gas for the qualitative analyses of compounds by positive ion gas chromatography–mass spectrometry (GCMS). The gas is readily available, inexpensive, and leaves no carbon contamination in the MS source. Compounds of interest to our laboratory typically yield abundant protonated or ammoniated species, which are indicative of a compound’s molecular weight. Nevertheless, some labile compounds fragment extensively by substitution and elimination reactions and yield no molecular weight information. In these cases, a CI reagent gas mixture of methylamine in methane prepared dynamically was found to be very useful in obtaining molecular weight data. Likewise, deuterated ammonia and deuterated methylamine are useful CI reagent gases for determining the exchangeable protons in organic compounds. Deuterated methylamine CI reagent gas is conveniently prepared by dynamically mixing small amounts of methylamine with excess deuterated ammonia.

Using direct infusion mass spectrometry for serum metabolomics in Alzheimer’s disease

Currently, there is no cure for Alzheimer’s disease and early diagnosis is very difficult, since no biomarkers have been established with the necessary reliability and specificity. For the discovery of new biomarkers, the application of omics is emerging, especially metabolomics based on the use of mass spectrometry. In this work, an analytical approach based on direct infusion electrospray mass spectrometry was applied for the first time to blood serum samples in order to elucidate discriminant metabolites. Complementary methodologies of extraction and mass spectrometry analysis were employed for comprehensive metabolic fingerprinting. Finally, the application of multivariate statistical tools allowed us to discriminate Alzheimer patients and healthy controls, and identify some compounds as potential markers of disease. This approach provided a global vision of disease, given that some important metabolic pathways could be studied, such as membrane destabilization processes, oxidative stress, hypometabolism, or neurotransmission alterations. Most remarkable results are the high levels of phospholipids containing saturated fatty acids, respectively, polyunsaturated ones and the high concentration of whole free fatty acids in Alzheimer’s serum samples. Thus, these results represent an interesting approximation to understand the pathogenesis of disease and the identification of potential biomarkers. Graphical Abstract

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Lipopolysaccharides of marine blue-green algae

The lipopolysaccharides (LPSs) have been isolated from two blue-green algae,Oscillatoria hilderbrandtii andNostoc sp. by the method of phenol-water extraction. The LPSs contain polysaccharide and lipid components. The polysaccharide fraction fromO. hildebrandtii consists predominantly of 1,3-bound rhamnose residues with a substituent in the second position. The rhamnose residues in the polysaccharide fraction ofNostoc are connected by 1,3- and 1,2-glycosidic bonds. The lipid components of the LPSs fromO. hilderbrandtii andNostoc consist of palmitic and steric acids, with glucosamine and glucose.     

Development of botanical and fish oil standard reference materials for fatty acids

As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60 % long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.

Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC–MS/MS

We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs ¹³C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®.

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

CO2 laser ionization of acoustically levitated droplets

For many analytical purposes, direct laser ionization of liquids is desirable. Several studies on supported droplets, free liquid jets, and ballistically dispensed microdroplets have been conducted, yet detailed knowledge of the underlying mechanistics in ion formation is still missing. This contribution introduces a simple combination of IR-MALDI mass spectrometry and an acoustical levitation device for contactless confinement of the liquid sample. The homebuilt ultrasonic levitator supports droplets of several millimeters in diameter. These droplets are vaporized by a carbon dioxide laser in the vicinity of the atmospheric pressure interface of a time of flight mass spectrometer. The evaporation process is studied by high repetition rate shadowgraphy experiments elucidating the ballistic evaporation of the sample and revealing strong confinement of the vapor by the ultrasonic field of the trap. Finally, typical mass spectra for pure glycerol/water matrix and lysine as an analyte are presented with and without the addition of trifluoracetic acid, and the ionization mechanism is briefly discussed. The technique is a promising candidate for a reproducible mass spectrometric detection scheme for the field of microfluidics.