Kristin Teuber

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

Significant sensitivity improvements by matrix optimization: a MALDI-TOF mass spectrometric study of lipids from hen egg yolk

Due to its sensitivity, the tolerance of impurities and the simplicity of performance, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to analyze lipids from biological sources. Although its detailed role is not understood so far, the applied matrix has a pronounced effect on the achievable spectrum quality and particularly how sensitive the individual lipid classes are detectable. Different matrix compounds were recently established in the lipid field including 2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9-AA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), and 2-(2-aminoethylamino)-5-nitropyridine (AAN). It is the aim of this paper to compare the properties of these matrices with the newly synthesized matrix, alpha-cyano-2,4-difluorocinnamic acid (Di-FCCA). An organic extract from hen egg yolk was used as a simple and easily available test system. It will be shown that Di-FCCA is the matrix of choice to detect lipids in the positive-ion mode due to an achievable sensitivity gain of more than one order of magnitude compared to alternative matrices. In contrast, Di-FCCA is not suitable for negative-ion detection of phospholipids. Here, 9-AA is unequivocally the matrix of choice.

The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31P NMR.

The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and (31)P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species. The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used.

Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel

AbstractHigh-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and 31P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations. FigurePhospholipids separated by HPTLC were subsequently re-eluted from the plate and characterized by different methods. Spectra in blue represent the original mixture that was applied onto the HPTLC plate, while the red spectra represent the phospholipids subsequent to HPTLC. Although there were significant losses in the amount of each phospholipid, no changes in the relative and fatty acyl compositions occurred.

2,4-Dinitrophenylhydrazine as a New Reactive Matrix to Analyze Oxidized Phospholipids by MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is increasingly used to analyze native and oxidized lipids. However, as the majority of MALDI devices are operating under high vacuum, the detection of volatile compounds such as “small” aldehydes is difficult and requires previous derivatization. This study uses 2,4-dinitrophenylhydrazine (DNPH) to efficiently convert aldehydes into hydrazones that can be easily identified by the characteristic mass shift and their selective enrichment by precipitation. Additionally, the UV absorption of the hydrazones at 337 nm is sufficiently high allowing direct analysis without typical MALDI matrices. Finally, it will be shown that the presence of DNPH does also enable the detection of non-oxidized phospholipids.

Characterization of oxidation products from 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine in aqueous solutions and their reactions with cysteine, histidine and lysine residues.

This report focuses on studies of lipid peroxidation products reactivity towards the side chains of cysteine, histidine, and lysine residues in structurally unordered peptides. Thus we have analyzed linoleic acid peroxidation products (LaPP) obtained by incubating 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) overnight with or without H(2)O(2) in the presence or absence of CuCl. In total, 55 different LaPP were identified with 26 containing reactive carbonyl groups. The strongest oxidation conditions (H(2)O(2) and Cu(I), i.e. a Fenton-like reagent) yielded 51 LaPP, whereas air oxidation produced only 12 LaPP. Independent of the oxidation conditions, around half of all LaPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition) PLPC oxidation products. The stronger oxidation conditions increased the number of LaPP, but also oxidized the added peptide Ac-PAAPAAPAPAEXTPV-OH (X=Cys, His or Lys) very quickly, especially under Fenton conditions. Thus, PLPC was oxidized by milder conditions (air or Cu(I)), incubated with the peptide and the peptide modifications were then analyzed by nano-RPC-ESI-Orbitrap-MS. Ten LaPP-derived peptide modifications were identified at lysine, whereas nine products were identified for cysteine and only three for histidine. Three high molecular weight LaPP still esterified to the GPC backbone were detected on Lys-containing peptide. Furthermore, three LaPP-derived mass shifts were obtained at cysteine, which have not previously been reported.

Chlorinated and brominated phosphatidylcholines are generated under the influence of the Fenton reagent at low pH-a MALDI-TOF MS study

Lipid (phospholipid) oxidation is an increasingly important research topic due to the significant physiological relevance. The Fenton reaction, i.e. the transition metal catalyzed decomposition of H(2)O(2) is frequently used to generate hydroxyl radicals (HO*). Lipids with unsaturated fatty acyl residues are primarily converted by HO* radicals into peroxides. In contrast, chloro- and bromohydrins as well as dihalogenides are formed by the addition of HOCl or HOBr to the olefinic groups of the fatty acyl residues of lipids or under the influence of the enzyme myeloperoxidase (MPO) from Cl(-) and H(2)O(2). We will show here by using MALDI-TOF MS for product analysis that halogenated products may also be generated in the presence of the Fenton reagent, if either FeCl(2) or FeBr(2) is used. In the presence of FeSO(4), however, peroxides are exclusively generated. It will also be shown that the generation of halogen-containing products is a competing reaction with the cleavage of the double bond under generation of the corresponding aldehyde or carboxylic acid that is favored at prolonged incubation times and at elevated pH.

MALDI-TOF mass spectrometry as a simple tool to determine the phospholipid/glycolipid composition of sperm: Pheasant spermatozoa as one selected example

Cellular membranes are composed of highly variable lipid molecules, mainly cholesterol and phospholipids (PLs). The cholesterol moiety and the saturation degree of the fatty acyl residues of PL determine the fluidity of the membrane, which is particularly important for sperm because they have to undergo characteristic membrane-dependent processes (acrosomal exocytosis and fusion with the oocyte). Glycolipids are an essential part of the membrane surface acting as key mediators in the interactions of sperm with components of the female genital tract. Although the lipid composition of many mammalian spermatozoa has already been determined, the lipid composition of avian spermatozoa has scarcely been investigated. Using spermatozoa extracts of the ring-necked pheasant (Phasianus colchicus) as a selected example, this work demonstrates that matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple and fast method to determine spermatozoal lipid compositions. The lipid compositions of pheasant spermatozoa have not yet been investigated. In addition to common membrane (primarily diacyl) PL (sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine), remarkable variation of different sulfoglycolipids (sulfogalactocerebrosides) was identified. This is in strong contrast to all other animal species investigated so far which nearly exclusively contain the sulfoglycolipid seminolipid (sulfogalactoalkylacylglycerol). We emphasize that the MALDI MS approach allows the characterization of sulfoglycolipids of sperm within a few minutes without the necessity for previous chromatographic separation.

TLC/HPTLC with Direct Mass Spectrometric Detection: A Review of the Progress Achieved in the Last 5 Years

Thin-layer chromatography (TLC) is a widely used, fast and inexpensive method. Even minor constituents of complex mixtures can be often characterized. Unfortunately, the unambiguous assignment of the spots to defined compounds is often difficult because common staining methods normally reveal only (at best) a substance class, not a specific compound. For instance, in the case of phospholipids, the lipid class, but not the detailed fatty acyl composition, can be determined (normal phase TLC). Nowadays mass spectrometry (MS) seems to be the most suitable method for analyte characterization due to its high sensitivity and mass accuracy. This became possible by the invention of soft ionization methods such as matrix-assisted laser desorption and ionization time-of-flight (MALDI–TOF) MS and electrospray ionization (ESI) although many further desorption techniques are available nowadays. This review summarizes the so far available knowledge about direct TLC/MS couplings and gives an overview about selected compounds that could be successfully analyzed.