On the stability of the organic dication of the bisquaternary ammonium salt decamethoxinum under liquid secondary ion mass spectrometry

In the course of a liquid secondary ion mass spectrometric (SIMS) investigation on a bisquaternary ammonium antimicrobial agent, decamethoxinum, unusual pathways of fragmentation of the organic dication M2+ of this bisquaternary salt, with preservation of the doubly charged state of the fragments, were observed. To reveal the structural and electronic parameters of decamethoxinum, which are responsible for the stabilization of its organic dication in the gas phase, a comprehensive SIMS study using metastable decay, collision-induced dissociation and kinetic energy release techniques complemented by ab initio quantum chemical calculations was performed. Pathways of fragmentation of two main precursors originating from decamethoxinum-organic dication M2+ and its cluster with a Cl- counterion [M.Cl]+-and a number of their primary fragments were established and systematized. Differences in the pathways of fragmentation of M2+ and [M.Cl]+ were revealed: the main directions of [M.Cl]+ decay involve dequaternization similar to thermal degradation of this compound, while in M2+ fragmentation via loss of one and two terminal radicals with preservation of the doubly charged state of the fragments dominates over charge separation processes. It was shown that pairing of the dication with a Cl- anion does not preserve the complex from fragmentation via separation of two positively charged centers or neutralization (dequaternization) of one such center. At the same time the low abundance of M2+ in the SIMS spectra is to a larger extent controlled by a probability of M2+ association with an anion than by the decay of the dication per se.Quantum chemical calculations of the structural and electronic parameters of the decamethoxinum dication have revealed at least three features which can provide stabilization of the doubly charged state. Firstly, in the most energetically favorable stretch conformation the distance between the quaternary nitrogens (rN1-N2=1.39 nm) is relatively large. Secondly, an intramolecular solvation of quaternary groups by carbonyl oxygens of the adjacent groups of the dication occurs, which contribute to structural stabilization. Thirdly, an important feature of the electronic structure of the dication is the presence of a partial negative charge on the nitrogen atoms and smearing of a positive charge mainly over the hydrogens of alkyl groups attached to the quaternary nitrogens, which reduces the net repulsion between the quaternary groups. The possible influence of charge smearing on the kinetic energy released on the dication fragmentation is discussed.     

Influence of molecular environment on the analysis of phospholipids by time-of-flight secondary ion mass spectrometry

Understanding the influence of molecular environment on phospholipids is important in time-of-flight secondary ion mass spectrometry (TOF-SIMS) studies of complex systems such as cellular membranes. Varying the molecular environment of model membrane Langmuir-Blodgett (LB) films is shown to affect the TOF-SIMS signal of the phospholipids in the films. The molecular environment of a LB film of dipalmitoylphosphatidylcholine (DPPC) is changed by varying the film density, varying the sample substrate, and the addition of cholesterol. An increase in film density results in a decrease in the headgroup fragment ion signal at a mass-to-charge ratio of 184 (phosphocholine). Varying the sample substrate increases the secondary ion yield of phosphocholine as does the addition of proton-donating molecules such as cholesterol to the DPPC LB film. Switching from a model system of DPPC and cholesterol to one of dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol demonstrates the ability of cholesterol to also mask the phospholipid headgroup ion signal. TOF-SIMS studies of simplistic phospholipid LB model membrane systems demonstrate the potential use of these systems in TOF-SIMS analysis of cells.     

Is there a ‘matrix suppression effect’ under fast‐atom bombardment liquid secondary ion mass spectrometry of ionic surfactants in glycerol?

Some features of a ‘matrix suppression effect’ caused by ionic surface‐active compounds under fast‐atom bombardment (FAB) liquid secondary ion mass spectrometry (LSIMS) are being revised. It is shown that abundant transfer of the glycerol matrix molecules to the gas phase does occur under FAB‐LSIMS of ionic surfactants, contrary to popular belief. This process can be obscure because of the dependence of the charge state of the glycerol‐containing cluster ions on the type of ionic surfactant. It is revealed that, while glycerol matrix signals are really completely suppressed in the positive ion mass spectra of cationic surfactants (decamethoxinum, aethonium), abundant deprotonated glycerol and glycerol‐anion clusters are recorded in the negative ion mode. In the case of an anionic surfactant (sodium dodecyl sulfate), on the contrary, glycerol is completely suppressed in the negative ion mode, but is present in the protonated and cationized forms in the positive ion mass spectra. It is suggested that such patterns of positive and negative ion FAB‐LSIMS spectra of ionic surfactants solutions reflect the structure and composition of the electric double layer formed at the vacuum‐liquid interface by organic cations or anions and their counterions. Processes leading to the formation of the glycerol‐containing ions preferentially of positive or negative charge are discussed. The most obvious of them is efficient binding of glycerol to inorganic counterions of the salts Cl^? or Na⁺, which is confirmed by data from quantum chemical calculations. The high content of the counterions and relatively small content of glycerol in the sputtered zone may be responsible for the charge‐selective suppression of neat glycerol clusters of opposite charge to the counterions. In the case of a mixture of cationic and anionic surfactants the substitution of inorganic counterions by organic ones was observed. The dependence of the exchange rate in the surface layer is not a linear function of the bulk solution concentration, and an effect of abrupt recharging of the surface can be registered. No both positively or negatively charged pure glycerol and glycerol‐inorganic counterion clusters are recorded for the mixture. Correlations between the mass spectrometric observations and some phenomena of surface and colloid chemistry and physics are discussed. Copyright © 2007 John Wiley & Sons, Ltd.     

Phosphatidylethanolamine-induced cholesterol domains chemically identified with mass spectrometric imaging

Coexisting liquid phases of model membrane systems are chemically identified using imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS). The systems studied were Langmuir-Blodgett (LB) model membranes of cholesterol (CH) with two different phospholipids, one a major component in the outer plasma membrane bilayer leaflet (dipalmitoylphosphatidylcholine (PC)) and the other a major component in the inner leaflet (dipalmitoylphosphatidylethanolamine (PE)). Binary mixtures of CH with each of the phospholipids were investigated, as well as a ternary system. A single homogeneous phase is evident for PC/CH, whereas both systems containing PE show lateral heterogeneity with phospholipid-rich and CH-rich regions. The interaction between CH and the two phospholipids differs due to the disparity between the phospholipid headgroups. Imaging TOF-SIMS offers a novel opportunity to chemically identify and differentiate the specific membrane locations of CH and phospholipid in membrane regions without the use of fluorescent dyes. This unique imaging method has been used to demonstrate the formation of micrometer-size CH domains in phosphatidylethanolamine-rich systems and is further evidence suggesting that CH may facilitate transport and signaling across the two leaflets of the plasma membrane.     

Determination of alkyl benzyl and dialkyl dimethyl quaternary ammonium biocides in occupational hygiene and environmental media by liquid chromatography with electrospray ionisation mass spectrometry and tandem mass spectrometry

A new method for the simultaneous qualitative and quantitative determination of alkyl benzyl and dialkyl quaternary ammonium compounds (QACs) has been developed. Analysis is by reversed-phase high-performance liquid chromatography coupled with electrospray ionisation mass spectrometry. QACs are extremely amenable to the electrospray ionisation technique (limit of detection of BAC C12 homologue 3 ng ml(-1)). The selectivity of mass spectrometric detection allows simultaneous determination of benzyl and dialkyl dimethyl ammonium compounds. The method was successfully applied to the analysis of real samples (occupational hygiene sampling devices, products and swimming pool water). Structural information was obtained by MS-MS and cone voltage ion dissociation techniques. Ion dissociation enabled the structural elucidation of an unknown quaternary ammonium compound present in a commercial formulation.     

Correlation of Insulin‐Enhancing Properties of Vanadium‐Dipicolinate Complexes in Model Membrane Systems: Phospholipid Langmuir Monolayers and AOT Reverse Micelles

We explore the interactions of V^III‐, V^IV‐, and V^V‐2,6‐pyridinedicarboxylic acid (dipic) complexes with model membrane systems and whether these interactions correlate with the blood‐glucose‐lowering effects of these compounds on STZ‐induced diabetic rats. Two model systems, dipalmitoylphosphatidylcholine (DPPC) Langmuir monolayers and AOT (sodium bis(2‐ethylhexyl)sulfosuccinate) reverse micelles present controlled environments for the systematic study of these vanadium complexes interacting with self‐assembled lipids. Results from the Langmuir monolayer studies show that vanadium complexes in all three oxidation states interact with the DPPC monolayer; the V^III–phospholipid interactions result in a slight decrease in DPPC molecular area, whereas V^IV and V^V–phospholipid interactions appear to increase the DPPC molecular area, an observation consistent with penetration into the interface of this complex. Investigations also examined the interactions of V^III‐ and V^IV‐dipic complexes with polar interfaces in AOT reverse micelles. Electron paramagnetic resonance spectroscopic studies of V^IV complexes in reverse micelles indicate that the neutral and smaller 1:1 V^IV‐dipic complex penetrates the interface, whereas the larger 1:2 V^IV complex does not. UV/Vis spectroscopy studies of the anionic V^III‐dipic complex show only minor interactions. These results are in contrast to behavior of the V^V‐dipic complex, [VO₂(dipic)]^?, which penetrates the AOT/isooctane reverse micellar interface. These model membrane studies indicate that V^III‐, V^IV‐, and V^V‐dipic complexes interact with and penetrate the lipid interfaces differently, an effect that agrees with the compounds’ efficacy at lowering elevated blood glucose levels in diabetic rats.     

Measurement of the anchorage force between GPI-anchored alkaline phosphatase and supported membranes by AFM force spectroscopy

Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.     

An electrospray ionisation tandem mass spectrometric investigation of selected psychoactive pharmaceuticals and its application in drug and metabolite profiling by liquid chromatography/electrospray ionisation tandem mass spectrometry

A tandem mass spectrometric investigation of the collision-induced dissociation of five commonly prescribed psychoactive pharmaceuticals, risperidone, sertraline, paroxetine, trimipramine, and mirtazapine, and their metabolites has been carried out. Quadrupole ion trap mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the compounds under investigation and structural assignments of product ions were supported by quadrupole time-of-flight mass spectrometry. These fragmentation studies were then utilised in the development of a liquid chromatographic method to identify the drugs and their metabolites in human hair and saliva samples, thus providing relevant profiling information.     

Interaction between amphiphilic peptides and phospholipid membranes

This brief review aims at providing some illustrative examples on the interaction between amphiphilic peptides and phospholipid membranes, an area of significant current interest. Focusing on antimicrobial peptides, factors affecting peptide–membrane interactions are addressed, including effects of peptide length, charge, hydrophobicity, secondary structure, and topology. Effects of membrane composition are also illustrated, including effects of membrane charge, nature of the polar headgroup, and presence of cholesterol and other sterols. Throughout, novel insights on the importance of peptide adsorption density on membrane stability are emphasized, as is the correlation between peptide adsorption, peptide-induced leakage in model liposome systems, peptide-induced lysis of bacteria, and bacteria killing.     

Analysis of sulfonated compounds by reversed-phase ion-pair chromatography—Mass spectrometry with on-line removal of non-volatile tetrabutyl ammonium ion-pairing agents

Summary An ion-pair reversed-phase high performance liquid chromatography method with tetrabutyl ammonium salts as ion-pairing agents and mass spectrometric detection has been developed for sulfonated compounds with special focus on structural identification. A cation exchange suppressor cartridge placed between the UV detector and the ion source of the mass spectrometer completely removed the non-volatile ion-pairing agent resulting in excellent conditions for both electrospray ioniziation and atmospheric pressure chemical ioniziation. Peak broadening caused by the dead volume of the suppressor is negligible. The application range of this method is demonstrated by the structural identification of individual compounds within a complex mixture of model substances consisting of sulfonated aromatics, textile dyes and detergents with as many as four sulfonic acid groups, all largely differing from each other in their structural properties. The influence of the ion-pairing agent's counter ion on retention behaviour is also discussed.     

Mass spectrometric studies on cyclo- and poly-phosphazenes: 4—Attempts at mass spectrometrically induced polymerization of cyclotriphosphazenes substituted with primary and secondary amines

In this paper, the mass spectrometric behaviour of cyclophosphazenes substituted with primary and secondary amines is investigated, together with the mass spectrometric polymerization pattern of these compounds. While it is not possible to achieve the mass spectrometric polymerization of hexa(anilino)cyclotriphosphazene, ion–molecule reactions take place in the case of hexa(piperidino)- and hexa(morpholino)cyclotriphosphazenes, which lead to the formation of hexameric species. Evidence is given that pentasubstituted cyclophosphazenephosphorus cations are the species responsible for the oligomerization process.     

Threshold dissociation energies of protonated amine/polyether complexes in a quadrupole ion trap

Electrospray ionization mass spectrometry (ESI-MS) is increasingly used to probe the nature of noncovalent complexes; however, assessing the relevance of gas-phase results to structures of complexes in solution requires knowledge of the types of interactions that are maintained in a solventless environment and how these might compare to key interactions in solution. This study addresses the factors impacting the strength of hydrogen bonding noncovalent interactions in the gas phase. Hydrogen bonded complexes consisting of ammonium ions bound to polydentate ethers are transported to the gas phase with ESI, and energy-variable collisional activated dissociation (CAD) is used to map the relative dissociation energies. The measured relative dissociation energies are correlated with the gas-phase basicities and steric factors of the amine and polyether constituents. To develop correlations between hydrogen bonding strength and structural features of the donor and acceptor molecules, a variety of amines with different gas-phase basicities and structures were selected, including primary, secondary, and tertiary amines, as well as those that are bidentate to promote intramolecular hydrogen bonding. The acceptor molecules are polydentate ethers, such as 18-crown-6. Four primary factors influence the observed dissociation energies of the polyether/ammonium ion complexes: the gas-phase basicities of the polyether and amine, steric effects of the amines, conformational flexibility of the polyethers, and the inhibition of intramolecular hydrogen bonds of the guest ammonium ions in the resulting ammonium/polyether noncovalent complexes.     

Photochemical activities of plant photosystem I particles reconstituted into phosphatidylglycerol liposomes

Phosphatidylglycerol (PG) is the only anionic phospholipid in photosynthetic membrane and the important component of photosystem I (PSI). In this study, the interaction of PG with PSI particle from spinach was investigated by using reconstitution method. The results from the properties of electron transport, fluorescence emission, turbidity, and protein secondary structures in PSI complex incorporated into PG liposomes revealed the existence of PSI-PG interactions. A stimulation and an inhibition of oxygen uptake in PSI particle at a low and higher PG/chlorophyll mass ratio, respectively, were observed. Moreover, an additional enhancement and depression of electron flow in the PSI-PG complexes were occurred in the reaction medium containing CaCl2 at concentrations below and above 5 mM, the aggregation threshold of the reconstituted membranes, respectively. The results demonstrated that the maintenance of the structural optimization was needed for a stimulation of electron transport at a low PG/PSI mass ratio, while a decay of this PSI activity at high PG/PSI ratio was the result of inhibition of the energy transfer from LHCI to PSI reaction center induced by the dissociation of LHCI-680.     

Conformational Space of a Polyphilic Molecule with a Fluorophilic Side Chain Integrated in a DPPC Bilayer

We investigate the conformational space of a polyphilic molecule with hydrophilic, lipophilic and fluorophilic parts inserted as a transmembrane agent into a dipalmitoylphosphatidylcholine bilayer by means of all‐atom molecular dynamics simulations. Special focus is put on the competing structural driving forces arising from the hydrophilic, lipophilic and fluorophilic side chains and the aromatic backbone of the polyphile. We observe a significant difference between the lipophilic and the fluorophilic side chains regarding their intramembrane distribution. While the lipophilic groups remain membrane‐centered, the fluorophilic parts tend to orient toward the phosphate headgroups. This trend is important for understanding the influence of polyphile agents on the properties of phospholipid membranes. From a fundamental point of view, our computed distribution functions of the side chains are related to the interplay of sterical, enthalpic and entropic driving forces. Our findings illustrate the potential of rationally designed membrane additives which can be exploited to tune the properties of phospholipid membranes. © 2017 Wiley Periodicals, Inc.     

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.     

The role of nucleophile--electrophile interactions in the unimolecular and bimolecular gas-phase ion chemistry of peptides and related systems

This account describes the experimental tools (multi-stage mass spectrometric experiments, isotopic and structural labelling, kinetics and theoretical modelling) and physical organic concepts (influence of charge, the intermediacy of ion-molecule complexes, etc.) that can be used to unravel the mechanisms of gas-phase unimolecular and bimolecular ionic reactions of peptides. The role that nucleophile-electrophile interactions play in charge-directed reactions is highlighted for both unimolecular fragmentations (examples are illustrated for protonated sulfur-containing amino acids and peptides) and bimolecular ion-molecule reactions which cleave peptide bonds.     

Drug partition chromatography on immobilized porcine intestinal brush border membranes

We immobilized porcine intestinal brush border membrane vesicles (BBMVs) for chromatographic analyses of drug partitioning into the membranes determined as Ks, the drug retention per phospholipid amount. For positive and neutral drugs Ks decreased day by day, whereas Ks for negative drugs increased marginally. Similar results on vesicle-lipid liposomes indicated a gradual loss of negative charge from the columns. The Ks values for positive drugs were higher than those for negative drugs with the same octanol/water partitioning or the same Ks on egg yolk phospholipid bilayers. Electrostatic interactions seem to be important for the partitioning of charged drugs into brush border membranes.     

Interactions between antimicrobial polynorbornenes and phospholipid vesicles monitored by light scattering and microcalorimetry

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.     

Enantiospecific interactions between cholesterol and phospholipids

The effects of cholesterol on various membrane proteins have received considerable attention. An important question regarding each of these effects is whether the cholesterol exerts its influence by binding directly to membrane proteins or by changing the properties of lipid bilayers. Recently it was suggested that a difference in the effects of natural cholesterol and its enantiomer, ent-cholesterol, would originate from direct binding of cholesterol to a target protein. This strategy rests on the fact that ent-cholesterol has appeared to have effects on lipid films similar to those of cholesterol, yet fluorescence microscopy studies of phospholipid monolayers have provided striking demonstrations of the enantiomer effects, showing opposite chirality of domain shapes for phospholipid enantiomer pairs. We observed the shapes of ordered domains in phospholipid monolayers containing either cholesterol or ent-cholesterol and found that the phospholipid chirality had a great effect on the domain chirality, whereas a minor (quantitative) effect of cholesterol chirality could be observed only in monolayers with racemic dipalmitoylphosphatidylcholine. The latter is likely to derive from cholesterol-cholesterol interactions. Accordingly, cholesterol chirality has only a modest effect that is highly likely to require the presence of solidlike domains and, accordingly, is unlikely to play a role in biological membranes.