Jürgen Schiller

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 suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound.

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.

Effects of hypochlorous acid on unsaturated phosphatidylcholines.

Effects of hypochlorous acid and of the myeloperoxidase-hydrogen peroxide-chloride system on mono- and polyunsaturated phosphatidylcholines were analyzed by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Chlorohydrins and glycols were detected as main products according to the characteristic shift of molecular masses. Mainly mono-chlorohydrins result upon the incubation of HOCl/(-)OCl with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, whereas only traces of mono-glycols were detected. 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine yielded a complex mixture of products. Mono-chlorohydrins and glycols dominated only at short incubation, while bis-chlorohydrins as well as products containing one chlorohydrin and one glycol moiety appeared after longer incubation. Similarly, a complex product mixture resulted upon incubation of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine with hypochlorous acid. Additionally, tris-chlorohydrins, products with two chlorohydrin and one glycol moiety, as well as lysophosphatidylcholines and fragmentation products of the arachidonoyl side chain were detectable. Mono-chlorohydrins of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine were detected after the incubation of the latter phospholipid with the myeloperoxidase-hydrogen peroxide-chloride system at pH 6.0. These chlorohydrins were not observed in the absence of chloride, hydrogen peroxide, or myeloperoxidase as well as in the presence of methionine, taurine, or sodium azide. Thus, mono-chlorohydrins in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine produced by hypochlorous acid from the myeloperoxidase-hydrogen peroxide-chloride system can also be detected by means of MALDI-TOF MS.

Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N = 5. Using 2,5-dihydroxybenzoic acid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidylcholine (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectable phospholipids. For phosphatidylinositol-tris-phosphate (PIP3) in a mixture with PC, the limit is about 20 times higher than for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI from biological lipid mixtures prior to the application of MALDI-TOF MS.

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.

Experiments towards quantification of saturated and polyunsaturated diacylglycerols by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry

Abstract As recently shown, different physiologically relevant lipid classes can easily be analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI–TOF MS). In the present study the first application of MALDI–TOF for the quantitative analysis of diacylglycerols is described. It is shown that the use of a suitable reference sample enables the quantification of diacylglycerols up to the picomolar range. The best reproducibility of quantitative results for diacylglycerols was obtained using a matrix of 2,5-dihydroxybenzoic acid in ethylacetate and incorporation of an internal standard of the same lipid class. A moderate laser power was used, resulting in a very low extent of fragmentation, allowing a quantification by using solely the highest signal arising from sodium adduct formation of diacylglycerols. A linear correlation between peak intensity and lipid concentration over one order of magnitude was found. The applicability of this new technique for the analysis of other lipids like phosphatidylcholines is also discussed.

Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example

AbstractPhospholipids (PL) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As in the case of polar molecules, however, the careful selection of the matrix is crucial for optimum results. 9-Aminoacridine (9-AA) was recently suggested as the matrix of choice to analyze PL mixtures because of (a) the improved sensitivity and (b) the reduction of suppression effects compared to other matrices. However, the distinction of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the negative ion mode is obscured as PC is also detectable as –CH3+ ion if 9-AA is used as matrix. This may result in the erroneous assignment of PC as a PE species. Using an organic extract from hen egg yolk as example it will be shown that the contribution of PC must be taken into consideration if the negative ion mass spectra are used to evaluate the fatty acyl compositions of PE mixtures. 9-AA can as well be used in hyphenated thin-layer chromatography (TLC)-MALDI-TOF MS where PC and PE are chromatographically well separated for unequivocal assignments. FigureComparison of negative ion MALDI-TOF mass spectra of isolated 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE) using either DHB (blue) or 9-AA (red) as matrix. The spectra differ significantly as a function of the matrix used. In case of 9-AA, POPC is detectable as negative ion subsequent to the loss of a -CH3 group, which complicates peak assignments when complex mixtures are analyzed