MALDI-TOF MS to monitor the kinetics of phospholipase A2-digestion of oxidized phospholipids.

Free fatty acids (FFA) are released through phospholipase A2 (PLA2), which cleaves the fatty acyl residue at the sn-2 position of phospholipids (PL). During inflammatory diseases, reactive oxygen species (such as HOCl) lead to the formation of oxidatively modified PL (e.g., chlorohydrin generation). It is still widely unknown to which extent the oxidation of PL influences their digestibility by PLA2. Additionally, investigations on the impact of the position of the unsaturated fatty acyl residue (sn-1 versus sn-2 position) and modifications of the headgroup (for instance phosphatidylcholine (PC) versus phosphatidylethanolamine (PE)) are also lacking. Therefore, the aim of this study is the investigation of these aspects using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to elucidate the PL/lysophospholipid (LPL) ratios as measures of the PLA2 digestibility. We will show that oxidative modifications of PL by HOCl have a considerable impact on the PLA2 digestibility, i.e., oxidation of the unsaturated fatty acyl residues leads to a reduced digestibility of both PC and PE. Besides, it will be shown that MALDI MS is a convenient and reliable tool to investigate the related changes.

Stationary phase thickness determines the quality of thin-layer chromatography/matrix-assisted laser desorption and ionization mass spectra of lipids.

Normal phase thin-layer chromatography (NP TLC) is an established method of (phospho)lipid analysis. The determination of the fatty acyl composition is, however, a more challenging task by NP TLC. The direct coupling of TLC separation with mass spectrometric detection (e.g., matrix-assisted laser desorption/ionization mass spectrometry, MALDI MS), however, enables a detailed characterization of complex lipid mixtures. Here we show that the thickness of the silica gel layer has a considerable effect on the quality of the mass spectra recorded directly from the TLC plate. In particular, the intensity of the matrix background signals can be reduced if "thinner" TLC layers are used.

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.

A simple method to identify ether lipids in spermatozoa samples by MALDI-TOF mass spectrometry.

Plasmalogens (alkenylacyl glycerophospholipids) are important lipid constituents of many tissues and cells (e.g., selected spermatozoa). Since the molecular weights of plasmalogens overlap with that of diacyl- or alkyl acyl lipids, sophisticated mass spectrometry (MS; including MS/MS) analysis is normally used for the unequivocal identification of plasmalogens. We will show here that a simple matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (without MS/MS capability) in combination with acidic hydrolysis and subsequent derivatization with 2,4-dinitrophenylhydrazine (DNPH) and/or digestion with phospholipase A2 (PLA2) is sufficient to determine the contributions of ether lipids in spermatozoa extracts. As neither diacyl nor alkylacyl lipids are sensitive to acids and do not react with DNPH, the comparison of the mass spectra before and after treatment with acids and/or DNPH addition readily provides unequivocal information about the plasmalogen content. Additionally, the released aldehydes are readily converted into the 2,4-dinitrophenylhydrazones and can be easily identified in the corresponding negative ion mass spectra. Finally, PLA2 digestion is very useful in confirming the presence of plasmalogens. The suggested method was validated by analyzing roe deer, bovine, boar, and domestic cat spermatozoa extracts and comparing the results with isolated phospholipids.

Phosphatidylcholine dimers can be easily misinterpreted as cardiolipins in complex lipid mixtures: a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric study of lipids from hepatocytes.

The liver is an important organ that is particularly involved in the lipid metabolism of the organism. Thus, high interest is nowadays focused on the lipid composition of the liver and particularly the liver parenchymal cells, the hepatocytes. Hepatocytes contain common phospholipids (PL) such as phosphatidylcholines, -ethanolamines and -inositols, for instance, that can be easily analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) even without previous separation of the PL mixture. However, in addition to common PL, hepatocytes possess also significant amounts of cardiolipin (CLP). The MS analysis of this PL is quite challenging because it (a) has a higher mass than common lipids and (b) possesses a higher negative charge. We will show here that caution is required if CLP is analyzed directly from the total lipid extract because PC dimers may be interpreted as cardiolipins if the positive ion MALDI mass spectra are analyzed.

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.

Phospholipids (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.

The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, (31)P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition.

BACKGROUND: Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs) from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. RESULTS: Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. CONCLUSION: The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm.

Comparison of the positive and negative ion electrospray ionization and matrix-assisted laser desorption ionization-time-of-flight mass spectra of the reaction products of phosphatidylethanolamines and hypochlorous acid.

Phosphatidylethanolamines (PEs) react with HOCl under formation of the mono- and dichloramines which are easily converted into secondary products (nitriles and imines). PEs with unsaturated acyl residues also give chlorhydrines. The aim of this study was to investigate whether all products may be detected by electrospray ionization (ESI) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Results indicated that chloramines and imines are nearly exclusively detectable by ESI-MS, whereas all other products are detectable by both MALDI and ESI-MS. Therefore, ESI-MS is superior for the detection of chlorinated products of PEs.

The reaction between phosphatidylethanolamines and HOCl investigated by TLC: fading of the dye primuline is induced by dichloramines.

Phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs) are abundant lipid constituents of the membranes of Escherichia coli. The reaction between these lipids and hypochlorous acid (HOCl), an important constituent of disinfectants, was investigated by combined thin-layer chromatography (TLC), mass spectrometry (MS), UV and fluorescence spectroscopy. Primuline is a common dye in lipid research that binds non-covalently to lipids and allows, thus, the direct evaluation of TLC plates by MS. However, primuline staining of the products between PE and HOCl is accompanied by fading of the dye. This only holds if acidic but not alkaline conditions are applied. Using a combination of TLC, UV and fluorescence spectroscopy, it will be shown that dichloramines of PE are responsible for the observed primuline fading. Since dichloramines are slowly converted under alkaline conditions into the nitriles that lack the characteristic UV properties of dichloramines, fading is not observed under alkaline conditions.

Analysis of stem cell lipids by offline HPTLC-MALDI-TOF MS.

MALDI-TOF MS is traditionally used for "proteomics", but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed.

Analysis of brain lipids by directly coupled matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance thin-layer chromatography.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed.

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.

The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC.

The lipid composition of algae is crucial for numerous structural and physiological aspects, e.g. the integrity of the photosynthetic complexes and the functionality of membrane-embedded processes as the photosynthetic electron transport in thylakoids or the mitochondrial respiration. In this paper the lipid composition of the organic extracts of the green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana are compared by using matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with thin-layer chromatography (TLC). The combined methods enable quantitative evaluation of the individual lipid classes as well as the determination of the relative acyl compositions. It will be shown that both algae differ in (a) the lipid classes, (b) the relative contribution of the individual lipid classes and (c) the acyl compositions. Differences in the acyl composition concern particularly the mono- and digalactosyl diacylglycerols. Glycerol-trimethylhomoserine and phosphatidylethanolamine are exclusively detected in the C. reinhardtii extracts, whereas phosphatidylcholine is a characteristic lipid of C. meneghiniana. Furthermore, the proportion of the acidic lipids sulfoquinovosyl-diacylglycerol and phosphatidylglycerol is significantly higher in the diatom than in C. reinhardtii.

MALDI-TOF MS of phosphatidylethanolamines: different adducts cause different post source decay (PSD) fragment ion spectra.

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly applied to lipids. However, positional acyl chain analysis of lipids by MALDI was so far scarcely described. In this paper, the fragmentation behavior of phosphatidylethanolamine (PE) is investigated by using post-source decay (PSD) MS. In dependence on the investigated adduct, significant differences could be obtained. It will be shown that in particular the negative ion spectra enable the determination of the individual acyl chains as well as their positions (sn-1 or sn-2). Therefore, MALDI-TOF PSD spectra are a real alternative to more sophisticated MS/MS methods.

Combined Use of MALDI-TOF Mass Spectrometry and 31P NMR Spectroscopy for Analysis of Phospholipids.

Lipids are important and abundant constituents of all biological tissues and body fluids. In particular, phospholipids (PL) constitute a major part of the cellular membrane, play a role in signal transduction, and some selected PL are increasingly considered as potential disease markers. However, methods of lipid analysis are less established in comparison to techniques of protein analysis. Mass spectrometry (MS) is an increasingly used technique to analyze lipids, especially in combination with electrospray ionization (ESI) MS which is the so far best established ionization method. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has itself proven to be also useful in the field of lipid analysis. 31P nuclear magnetic resonance (NMR) spectroscopy is another powerful method of PL analysis, represents a direct quantitative method, and does not suffer from suppression effects.This chapter gives an overview of methodological aspects of MALDI-TOF MS and 31P NMR in lipid research and summarizes the specific advantages and drawbacks of both methods. In particular, suppression effects in MS will be highlighted and possible ways to overcome this problem (use of different matrices, separation of the relevant lipid mixture prior to analysis) will be discussed.

Nitrite reduces the effects of HOCl on unsaturated phosphatidylcholines--a MALDI-TOF MS study.

The concentration of nitrite (NO2-) increases under inflammatory conditions. However, the physiological role of nitrite is so far controversial discussed: it was reported that effects of HOCl (an important inflammation mediator) on phospholipids (PL) may be enhanced but also reduced in the presence of nitrite. In this paper a simple model system was used: unsaturated phosphatidylcholine (PC) vesicles were treated with HOCl in the presence of varying NaNO2 concentrations and the yield of reaction products was determined by MALDI-TOF MS: the extent of chlorohydrin generation was significantly reduced in the presence of NaNO2 because HOCl is consumed by the oxidation of NO2- to NO3-. Similar results were obtained when HOCl was generated by the myeloperoxidase (MPO)/H2O2/Cl- system or the experiments were carried out in the presence of a simple peptide. It is concluded that the transient products of the reaction between HOCl and NO2- do not have a sufficient reactivity to modify PL.

Analysis of the phospholipid composition of bronchoalveolar lavage (BAL) fluid from man and minipig by MALDI-TOF mass spectrometry in combination with TLC.

Surfaces of lungs are covered by the surfactant, an aqueous mixture of different phospholipids (PL) and proteins. Although the surfactant represents a relatively simple mixture of only a few PL (primarily phosphatidylcholine (PC) and phosphatidylglycerol (PG)), reliable methods of routine lipid analysis of the surfactant are still lacking. It will be shown that matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) represents a suitable technique for the differentiation of the apolar components of the surfactant of different species. Samples of man and minipig are used in this study since both are known to vary in their PL composition. PL of surfactant were separated by thin-layer chromatography (TLC) and the obtained subfractions subjected to MALDI-TOF MS analysis in order to monitor the presence of even minor PL species. It will be shown that besides PG and PC, also phosphatidylethanolamine, -inositol and sphingomyelin can be detected in surfactant of man, whereas only sphingomyelin could be detected in the minipig sample.

Lipid analysis by thin-layer chromatography--a review of the current state.

High-performance thin-layer chromatography (HPTLC) is a widely used, fast and relatively inexpensive method of separating complex mixtures. It is particularly useful for smaller, apolar compounds and offers some advantages over HPLC. This review gives an overview about the special features as well as the problems that have to be considered upon the HPTLC analysis of lipids. The term "lipids" is used here in a broad sense and comprises fatty acids and their derivatives as well as substances related biosynthetically or functionally to these compounds. After a short introduction regarding the stationary phases and the methods how lipids can be visualized on an HPTLC plate, the individual lipid classes will be discussed and the most suitable solvent systems for their separation indicated. The focus will be on lipids that are most abundant in biological systems, i.e. cholesterol and its derivates, glycerides, sphingo- and glycolipids as well as phospholipids. Finally, a nowadays very important topic, the combination between HPTLC and mass spectrometric (MS) detection methods will be discussed. It will be shown that this is a very powerful method to investigate the identities of the HPTLC spots in more detail than by the use of common staining methods. Future aspects of HPTLC in the lipid field will be also discussed.

An update of MALDI-TOF mass spectrometry in lipid research.

Although matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS)--often but not exclusively coupled with a time-of-flight (TOF) mass analyzer--is primarily established in the protein field, there is increasing evidence that MALDI MS is also very useful in lipid research: MALDI MS is fast, sensitive, tolerates sample impurities to a relatively high extent and provides very simple mass spectra without major fragmentation of the analyte. Additionally, MALDI MS devices originally purchased for "proteomics" can be used also for lipids without the need of major system alterations. After a short introduction into the method and the related ion-forming process, the MALDI mass spectrometric characteristics of the individual lipid (ranging from completely apolar hydrocarbons to complex glycolipids with the focus on glycerophospholipids) classes will be discussed and the progress achieved in the last years emphasized. Special attention will be paid to quantitative aspects of MALDI MS because this is normally considered to be the "weak" point of the method, particularly if complex lipid mixtures are to be analyzed. Although the detailed role of the matrix is not yet completely clear, it will be also explicitly shown that the careful choice of the matrix is crucial in order to be able to detect all compounds of interest. Two rather recent developments will be highlighted: "Imaging" MS is nowadays widely established and significant interest is paid in this context to the analysis of lipids because lipids ionize particularly well and are, thus, more sensitively detectable in tissue slices than other biomolecules such as proteins. It will also be shown that MALDI MS can be very easily combined with thin-layer chromatography (TLC) allowing the spatially-resolved screening of the entire TLC plate and the detection of lipids with a higher sensitivity than common staining protocols.