Marijana Petković

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.

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.

CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures 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) is mainly used for protein and peptide analysis. However, there is growing evidence that also phospholipids like phosphatidylcholines (PC) can be easily analyzed by MALDI-TOF MS. In MALDI-TOF methodology, the sample is cationized by the addition of inorganic ions. This process is strongly dependent on the corresponding ion concentration. In biological samples various cations are present (mainly H+, Na+ and K+) and, therefore, a mixture of different adducts is formed. Since phospholipids exhibit a wide distribution of different fatty acid residues a considerable peak overlap may occur. This is a major problem since the peak assignment in a mixture will be often unclear. In this paper we demonstrate that this problem can be easily overcome by mixing the analyte with caesium chloride (CsCl). This yields naturally non-occurring Cs+ adducts that are apparent due to the large shift of the molecular mass. The proposed method facilitates the clear assignment of most peaks. Besides that, we will show that CsCl can also be used for the determination of the relative fatty acid composition of a given PC sample. For this purpose naturally occurring mixtures of PCs as well as organic extracts of human lipoproteins-that are mainly composed of PC and sphingomyeline-are used.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A2

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.

Effects of thermal stressing on saturated vegetable oils and isolated triacylglycerols - product analysis by MALDI-TOF mass spectrometry, NMR and IR spectroscopy

It is well-known that triacylglycerols in vegetable oils undergo slow oxidative modifications upon storage particularly at elevated temperatures. This has been shown primarily for oils with unsaturated fatty acid residues that are most sensitive towards oxidation. Saturated oils, however, were by far less investigated. In the present study saturated oils (coconut oil) as well as isolated triacylglycerols were exposed to defined thermal stressing and the resulting products were investigated in dependence on temperature and the heating period. Matrix-assisted laser desorption and ionization spectrometry, 13C and 31P nuclear magnetic resonance spectroscopy and infrared spectroscopy were used for the characterization of the native as well as the thermally stressed oil samples. These methods were used since they provide both, fast and reliable information on oil composition and can be performed faster than other more established methods. We found that the degradation mechanism of saturated fatty acids is completely different from unsaturated fatty acids. Whereas unsaturated oils are primarily depleted under the cleavage of the double bonds, saturated oils undergo a conversion of one methylene group into a carbonyl group. This was independently demonstrated by all applied methods for the triacylglycerols as well as for the free fatty acids derived after saponification.

The Photoprotein Pholasin As a Luminescence Substrate for Detection of Superoxide Anion Radicals and Myeloperoxidase Activity in Stimulated Neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

Involvement of phosphatidic acid in both degranulation and oxidative activity in fMet-Leu-Phe stimulated polymorphonuclear leukocytes.

Background/Aim: Polymorphonuclear leukocytes (neutrophils) release a variety of toxic agents – proteins and reactive oxygen species (ROS) – that are used to inactivate foreign microorganisms in the non-specific immune response. This study was undertaken to compare intracellular signalling pathways that lead to the ROS production as well as degranulation of azurophilic granules of human fMet-Leu-Phe/cytochalasin B stimulated neutrophils. Methods: Luminol-amplified chemiluminescence was used for monitoring the oxidative activity of human neutrophils in the presence of various inhibitors. The elastase activity was assessed in the neutrophil supernatant as a marker for degranulation of azurophilic granules. Results: Tested inhibitors of enzymes of signalling cascades showed the same effect on the ROS production and on the activity of elastase released from neutrophils. The only difference was obtained with staurosporine: it inhibited the chemiluminescence response, but increased the elastase release. Conclusion: Early signalling pathways leading to the ROS production and the degranulation are ubiquitous in human neutrophils. They are branching most probably at the point of the phosphatidic acid production by phospholipase D. A protein kinase activated by this lipid second messenger might play a central regulatory role in human neutrophils.

Colloidal TiO2 nanoparticles as substrates for M(S)ALDI mass spectrometry of transition metal complexes

RATIONALE Nanoparticles as substrates for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have advantages over organic matrices, since they enable acquisition of spectra in the low-mass range. It has been previously shown that TiO(2) nanoparticles can be used as substrate for MALDI-TOF MS analysis of phospholipids and for other types of molecules, but none of them was applied to the analysis of transition metal complexes. METHODS The MALDI-TOF mass spectra of potential anti-tumor drugs [AuCl(2)(bipy)]Cl, [PtCl(4)(bipy)], and [RuCl(2)(bipy)(2) ]Cl acquired with organic matrices have been compared with spectra acquired with colloidal titanium dioxide nanoparticles. Colloidal TiO(2) nanoparticles (NPs) with average diameter of 5 nm were synthesized and characterized by microscopy. For some experiments, the TiO(2) NPs were treated at 60 °C. Suspensions of matrix and the analyte were premixed, applied to the MALDI target and left at room temperature. Mass spectra were acquired with a 50-Hz pulsed nitrogen laser emitting at a wavelength of 337 nm. RESULTS The MALDI spectra of transition metal complexes acquired with TiO(2) NPs exhibited somewhat lower sensitivity than those with organic matrices; on the other hand, they are characterized by significantly lower number of signals arising from the tested complexes than the organic matrices. Whereas adducts between organic matrices and the analytes were detectable in the spectra, this was not the case for the TiO(2)-assisted mass spectra. CONCLUSIONS We have shown that colloidal TiO(2) NPs can be used as substrates for MALDI-TOF MS of transition metal complexes. Although the sensitivity of this approach in comparison with the use of organic matrices might still be a problem, the potential of the applications of NPs for the mass spectrometric characterization of transition metal complexes is clearly demonstrated.

Application of flavonoids -quercetin and rutin -as new matrices for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of Pt(II) and Pd(II) complexes

Attempts are being made to overcome the resistance of tumour cells to platinum (Pt) drugs by the synthesis of new generations of Pt complexes, and it is important to find appropriate and simple methods for the characterization of those novel complexes. The additional applicability of such a method for the analysis of the interactions of metal complexes with biomolecules would be advantageous. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) seems to possess the capability to become this method of choice, since it could be applied to low-mass complexes as well as for the analysis of large biomolecules. In this work the applicability of flavonoids - quercetin and rutin - as matrices for MALDI-TOFMS analysis of dichlorido(ethylendiamine)platinum(II) ([PtCl(2)(en)]), dichlorido(diaminocyclohexane)platinum(II) ([PtCl(2)(dach)]) and chloride (diethylenetriamine) palladium(II) chloride ([PdCl(dien)]Cl) complexes is demonstrated. Spectra of Pt(II) and Pd(II) complexes recorded in the presence of quercetin and rutin are rather simple: Pt(II) complexes generate [M+Na](+) or [M+K](+)ions, whereas the investigated Pd(II) complex gives ions generated by the loss of one Cl(-) or HCl. Flavonoids give a relatively small number of well-defined ions in the low-mass region (at m/z 303.3 for quercetin and m/z 633.5 for rutin). Quercetin and rutin can be applied in much lower concentrations than other common MALDI matrices and require rather low laser intensity. We speculate that flavonoids stabilize the structures of the metal complexes and that they may be useful for the analysis of other biologically active metal complexes, thus implying their broader applicability.

Na + ,K + -ATPase as the Target Enzyme for Organic and Inorganic Compounds

This paper gives an overview of the literature data concerning specific and non specific inhibitors of Na+,K+-ATPase receptor. The immobilization approaches developed to improve the rather low time and temperature stability of Na+,K+-ATPase, as well to preserve the enzyme properties were overviewed. The functional immobilization of Na+,K+-ATPase receptor as the target, with preservation of the full functional protein activity and access of various substances to an optimum number of binding sites under controlled conditions in the combination with high sensitive technology for the detection of enzyme activity is the basis for application of this enzyme in medical, pharmaceutical and environmental research.