► Certain lipid species are increasingly considered to be of therapeutic value. ► Separation is an important prerequisite for successful lipid analysis. ► As MALDI-MS makes use of a solid sample ...combined TLC–MALDI-MS seems straightforward. ► Useful TLC/MALDI-MS matrices and application methods are discussed. ► Phospholipids and glycolipids can be successfully analyzed by TLC/MALDI-MS.
Thin-layer chromatography–matrix-assisted laser desorption and ionization mass spectrometry (TLC–MALDI-MS) of organic extracts from biological samples allows untargeted analysis and structural characterization of phospholipids and glycolipids ionized from the near-surface region of a sample separated on a TLC-plate. In particular, MALDI-MS enables the sensitive detection of many analytes directly from the solid surface of an ordinary TLC-plate even without previous staining. It will be shown that the detailed fatty acyl composition of phospholipids can be determined solely by TLC–MALDI-MS without previous derivatization, enzymatic cleavage and/or reversed phase separation. MALDI-time-of-flight (TOF) MS is thus a powerful method in this field due to its high sensitivity, low extent of induced fragmentation and simple, user-friendly performance. This review summarizes the so far available knowledge about combined TLC–MALDI-MS for phospholipid and glycolipid characterization together with the technical workflow and a survey of applications. Finally a perspective on the future of TLC–MALDI-MS is given.
Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the ...release of phospholipases particularly phospholipase A
2
(PLA
2
) and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA
2
leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively.
Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI.
Figure
Scheme of mass spectrometric analysis to study oxidation and enzyme-modified phospholipids changed under inflammatory conditions
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.
Phospholipids (PL) are converted into lipid biomarkers by the action of phospholipases and reactive oxygen species (ROS), which are activated or released under certain physiological and ...pathophysiological conditions. Therefore, the
in vivo
concentration of such lipid biomarkers e.g., lysophospholipids (LPLs) is altered in humans and animals under different conditions such as inflammation, stress, medication, and nutrition. LPLs are particularly interesting because they are known to possess pro- and anti-inflammatory properties and may be generated by two different pathways: either by the influence of phospholipase A
2
or by different reactive oxygen species that are generated in significant amounts under inflammatory conditions. Both lead to the cleavage of unsaturated acyl residues. This review provides a short summary of the mechanisms by which lipid biomarkers are generated under
in vitro
and
in vivo
conditions. The focus will be on lysophosphatidylcholine (LPC) because usually, this is the LPL species which occurs in the highest concentration and is, thus, easily detectable by chromatographic and spectroscopic methods. Finally, the effects of lipid biomarkers as signaling molecules and their roles in different human and animal pathologies such as infertility, cancer, atherosclerosis, and aging will be shortly discussed.
Application of MALDI-TOF mass spectrometry in lipidomics Fuchs, Beate; Schiller, Jürgen
European journal of lipid science and technology,
2009, 2009-01, No. 1 January 2009, 2009-01-00, Letnik:
111, Številka:
1
Journal Article
Recenzirano
Lipids are of high interest on different levels. First, lipids as triacylglycerols (TAG) (particularly different vegetable oils) are of immense commercial interest. Second, lipids are of high ...relevance for different in vivo processes. For instance, the diagnostics of changes of the lipoproteins of human blood is very important in vascular diseases, whereas phospholipids (PL) are crucial for the properties of the cellular membrane and some selected PL were also recognized to represent important second messengers. Although there are many different methods (including different chromatographic and spectroscopic approaches) available, mass spectrometry (MS) is the most versatile tool of lipid analysis, with or without prior separation steps. Although several different ionization techniques are capable of producing ions from the lipids of interest, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS is the simplest and most convenient method. This review gives a short methodological survey of the capabilities and drawbacks of MALDI-TOF MS and discusses its role in lipidomic studies. Although a summary about PL analysis will also be given, the focus of this review is the analysis of apolar lipids as TAG, cholesterol and cholesteryl esters. Aspects of sample preparations and the avoidance of pitfalls are also highlighted. Finally, it is the aim of this paper to show that MALDI MS provides data comparable to other modern MS methods, but in a faster and more convenient way.
Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular ...dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N6-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.
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► Inducible nitric oxide synthase (iNOS) loss protects mice from smoke-induced emphysema ► Pulmonary hypertension is dependent on iNOS activity in bone marrow-derived cells ► Emphysema is dependent on iNOS activity in non-BM-derived cells ► The iNOS inhibitor L-N6-(1-iminoethyl)-lysine improves established emphysema in mice
Smoking-related damage can be reversed by inhibition of iNOS.
Pulmonary hypertension (PH) is a life-threatening disease, characterized by pulmonary vascular remodeling. Abnormal smooth muscle cell proliferation is a primary hallmark of chronic hypoxia-induced ...PH. Essential for cell growth are alterations in the intracellular Ca(2+) homeostasis. Classical transient receptor potential (TRPC) proteins have been suggested to contribute to PH development, as TRPC1 and TRPC6 are predominantly expressed in precapillary pulmonary arterial smooth muscle cells (PASMC). Studies in a TRPC6-deficient mouse model revealed an essential function of TRPC6 in acute but not in chronic hypoxia.
We aimed to identify the importance of TRPC1 in the pathogenesis of chronic hypoxia-induced PH in mice.
TRPC1 expression analysis was performed using real-time polymerase chain reaction. TRPC1 function was assessed by in vivo experiments in TRPC1(-/-) animals as well as in isolated precapillary murine PASMC after TRPC1 knockdown by TRPC1-specific small interfering RNAs.
Only TRPC1 mRNA was up-regulated under hypoxia in isolated murine PASMC (1% O2 for 72 h). Hypoxia-induced proliferation of murine PASMC was attenuated in cells treated with small interfering RNA against TRPC1 and in cells isolated from TRPC1(-/-) animals compared with untreated and wild-type cells. TRPC1(-/-) mice did not develop PH in response to chronic hypoxia (FI(O2) 0.10 for 21 d) and had less vascular muscularization but a similar degree of right ventricular hypertrophy compared with wild-type mice.
Our results indicate an important role of TRPC1 in pulmonary vascular remodeling underlying the development of hypoxia-induced PH.
Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking ...nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(y/-)) or the classical transient receptor potential channel 6 (TRPC6(-/-)) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(2+) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(y/-) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.
Legionella pneumophila is the primary causative agent of Legionnaires’ disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the ...L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.
Although the most important application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is “proteomics,” there is growing evidence that this soft ...ionization method is also useful for phospholipid (PL) analysis. Although all PLs are detectable by MALDI-TOF MS, some lipid classes, particularly those with quaternary amines such as phosphatidylcholines (PCs), are more sensitively detected than others, and these suppress the signals of less sensitively detected PLs when complex mixtures are analyzed. Therefore, a separation of the total organic extract into individual lipid classes is necessary. As MALDI uses a solid sample, the direct evaluation of thin-layer chromatography (TLC) plates is possible. We report here on a method of directly coupling MALDI-TOF MS and TLC that can be easily implemented on commercially available MALDI-TOF devices. A total extract of hen egg yolk is used as a simple PL mixture to demonstrate the capabilities of this method. It will be shown that “clean” spectra without any major contributions from fragmentation products and matrix peaks can be obtained, and that this approach is even sensitive enough to detect the presence of PLs at levels of less than 1% of the total extract. graphic removed
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