Klaus Arnold

MALDI-TOF MS in lipidomics.

So far, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) seemed to be nearly a synonym for protein analysis. However, there is growing evidence that this technique is also an useful tool in lipid analysis and lipidomics because of its fast, simple and convenient performance allowing to record mass spectra of cells, crude tissue or body fluid extracts or even intact tissue slices in a few minutes. On the negative side, however, the reproducibility of MALDI-TOF mass spectra depends significantly on the homogeneity of the co-crystals between matrix and analyte and different lipid classes are detected with different sensitivities. This is especially important because lipids with quaternary ammonia groups (e.g., GPCho) may prevent the detection of other lipid classes (e.g., GPEtn). This review starts with a short overview on traditional methods of lipid analysis with the focus on mass spectrometric methods and compares MALDI-TOF MS with other important ionization techniques. Afterwards, some landmarks in the development of MALDI-TOF MS will be introduced and some important examples in the field of tissue and body fluid lipid analysis will be discussed. This review ends with a short outlook and summary focusing on the advantages and drawbacks of MALDI-TOF MS in lipidomics.

Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy - effects of freezing and thawing.

In the present study, the applicability of proton NMR spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to the analysis of the lipid composition of human spermatozoa and seminal fluids as well as changes after cryopreservation of human spermatozoa was investigated. Whereas NMR spectra primarily indicated a high content of double bonds within the spermatozoa but no marked differences upon cryopreservation, MS detected intense peaks which could be assigned to phosphatidylcholines containing one docosahexaenoic and one palmitic or stearic acid residue (m/z=806 and 834). In contrast, the seminal plasma contained more saturated fatty acids and especially more sphingomyelin (SM). A freezing/thawing cycle markedly influences the lipid composition of spermatozoa. There was a diminution of phosphatidylcholines (16:0, 22:6 and 18:0, 22:6) and SM (16:0) and the appearance of lysophosphatidylcholines (16:0 and 18:0) and ceramide (16:0). These data demonstrate the release or activation of both phospholipase A(2) and sphingomyelinase in human spermatozoa due to the freezing/thawing cycle. These results were finally confirmed by experiments on the action of phospholipases on lipids containing docosahexaenoic acid.

The dielectric properties of aqueous solutions of poly(ethylene glycol) and their influence on membrane structure

The dielectric constant of water is reduced drastically on addition of poly(ethylene glycol). The behaviour is not described by a linear mixture equation. The decreased dielectric constant can lead to the general perturbation of the membrane structure which is necessary in such a manner that a strong aggregation of membranes would lead to their fusion. The changed cation permeability in the presence of poly(ethylene glycol) can explained as the effect of the lowered dielectric constant on the transfer energy.

Dynamics of Membrane Penetration of the Fluorescent 7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl (NBD) Group Attached to an Acyl Chain of Phosphatidylcholine

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

Calorimetric, 13C NMR, and 31P NMR studies on the interaction of some phenothiazine derivatives with dipalmitoyl phosphatidylcholine model membranes.

1. The dipalmitoyl phosphatidylcholine/water system was employed to study the interaction of phenothiazines with model membranes. In particular the effects of the drugs upon the lipid phase transition were examined using differential scanning calorimetry and NMR spectroscopy. The studied phenothiazines have peripheral (diethazine) or central (chlorpromazine) properties. 2. Both drugs were observed to lower the phase transition temperature of dipalmitoyl phosphatidylcholine. The molar activity of chlorpromazine is somewhat higher than of diethazine. At low concentrations the drugs affect the dipalmitoyl phosphatidylcholine pretransition endotherm. 3. In the 13C NMR spectra of the drug-containing samples the signal of the trimethylammonium group of dipalmitoyl phosphatidylcholine is broadened, whereas a narrowing of the signal of the fatty-acid chain methylene groups is observed. Further, addition of the phenothiazines causes higher values of the effective chemical shift anisotropy of the 31P in the phosphate group, in comparison to the pure dipalmitoyl phosphatidylcholine sample. 4. The results obtained by three different techniques indicate a higher fluidity in the fatty-acid chain region and a mobility reduction of the polar headgroup of the dipalmitoyl phosphatidylcholine molecules in the presence of the phenothiazines. These phenomena can be well accounted for by a model for the incorporation of the phenothiazines in the dipalmitoyl phosphatidyl-choline bilayer, in which the dialkylaminoalkyl chains are located near the polar headgroups and the ring system does not penetrate far beyond the glycerol backbone into the hydrocarbon phase.

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of 2H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of 31P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.

Exclusion of poly(ethylene glycol) from liposome surfaces

The electrophoretic mobility of vesicles is measured for concentrations of poly(ethylene glycol) from 0 wt.% to 10 wt.%. Mixtures of phosphatidylcholines and phosphatidic acid are used. The zeta potential calculated from the electrophoretic mobility and the viscosity of the suspension becomes more negative for all vesicles studied. Binding of poly(ethylene glycol) to the phospholipid surface by addition of the poly(oxyethylene)-containing surfactant C12E8 has the opposite effect and a decrease of the zeta potential is observed. Independent measurements of the surface potential of the vesicles in the presence of PEG by use of a positively charged spin probe and ESR spectroscopy and a fluorescent pH indicator and fluorescence spectroscopy show that actually the surface potential is not changed. A theory of the electrophoretic behaviour of vesicles in the presence of PEG is given which explains the contradiction between the two methods. It is assumed that the polymer is excluded from the vesicle surface (depletion layer) and that the viscosity near the surface is lower than the viscosity in the bulk phase. The thickness of the depletion layer is calculated from the experiments. The decrease of the zeta potential in the presence of poly(oxyethylene) chains linked to the vesicle surface results from the friction increase.

Self-diffusion of water in cartilage and cartilage components as studied by pulsed field gradient NMR.

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self‐diffusion behavior of water molecules in cartilage, polymeric cartilage components, and different model polymers. The short‐time self‐diffusion coefficients (diffusion time Δ ≈ 13 msec) are found to decrease steadily with decreasing water content. This holds equally well for cartilage and cartilage components. The short‐time diffusion coefficients are subjected to a rather nonspecific obstruction effect and mainly depend on the water content of the sample. The long‐time diffusion coefficients in cartilage (Δ ≈ 500 msec), however, reflect structural properties of this tissue. Measurements with varying observation times as well as experiments involving enzymatic treatment of articular cartilage suggest that the collagenous network in cartilage is likely to be responsible for the observed restricted diffusion. Magn Reson Med 41:285–292, 1999.

Hydration of polymeric components of cartilage — an infrared spectroscopic study on hyaluronic acid and chondroitin sulfate

Hydrated polysaccharides are major constituents of cartilage and play an important role in its water-binding properties. Infrared (IR) spectroscopy and sorption isotherms have been used to investigate the hydration behavior of the glycosaminoglycans hyaluronic acid and chondroitin sulfate. IR-dichroism of the vibrational modes of the pyranose ring is found at relative humidities (RH) smaller than 84%. The IR-dichroism data for the vibrational modes of the pyranose ring have been analyzed with respect to the helical structure of these polysaccharides. The orientation vanishes at higher relative humidities (>84%), because a strong increase in the water uptake occurs in the observed sorption isotherms. Differences in the IR-absorbance of the O-H stretching mode of sorbed water between hyaluronic acid and chondroitin sulfate are shown to be caused by the additional hydration of the sulfate groups. The corresponding H-bonds are weaker than those of the hydration shell of the pyranose rings.

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization.

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.