Tandem mass spectrometric approach for determining structure of molecular species of aminophospholipids

Aminophospholipids, including glycerophosphatidylethanolamine, glycerophosphatidylserine and their Lyso analogous, have been analyzed by positive and negative ion liquid secondary ion ionization coupled to tandem mass spectrometry. The mass spectra of aminophospholipids obtained by tandem mass spectrometers with different configuration (liquid secondary ion-electric-magnetic sector coupled to quadrupole mass analyzer (low-energy collision) or electricmagnetic sector (high-energy collision), as well as electrospray ionization-quadrupole mass analyzer combined with quadrupole mass analyzer (low-energy collision), are compared. The mass spectra produced by low-energy collisionally induced dissociation of the deprotonated molecules from aminophospholipids contain fragment ions for characterizing polar head moieties as well as fatty acid composition and position. The mass spectra generated by high-energy collisionally induced dissociation of both protonated and deprotonated molecules from aminophospholipids show numerous product ions for identifying polar heads, composition, and location of fatty acid chains in molecular species. Triple quadrupole mass spectrometer with electrospray ionization exhibits remarkable superiority in detection sensitivity. Liquid secondary ion with electric-magnetic sector coupled to quadrupole mass analyzer or electric-magnetic sector instrument has the advantage of the capability of properly determining location of fatty acid chains in molecular species. This paper also describes an approach for structurally analyzing aminophospholipid species as 9-fluorenylmethyloxy-carbonyl derivatives by positive and negative ion liquid secondary ion mass spectrometry and high-energy collisionally induced dissociation tandem mass spectrometry. It has been found that the derivatives of glycerophosphatidylethanolamine and glycerophosphatidylserine can readily be analyzed by the negative ion liquid secondary ion and tandem mass spectrometric methods.     

From Supramolecular Hydrogels to Multifunctional Carriers for Biologically Active Substances

Supramolecular hydrogels are 3D, elastic, water-swelled materials that are held together by reversible, non-covalent interactions, such as hydrogen bonds, hydrophobic, ionic, host–guest interactions, and metal–ligand coordination. These interactions determine the hydrogels’ unique properties: mechanical strength; stretchability; injectability; ability to self-heal; shear-thinning; and sensitivity to stimuli, e.g., pH, temperature, the presence of ions, and other chemical substances. For this reason, supramolecular hydrogels have attracted considerable attention as carriers for active substance delivery systems. In this paper, we focused on the various types of non-covalent interactions. The hydrogen bonds, hydrophobic, ionic, coordination, and host–guest interactions between hydrogel components have been described. We also provided an overview of the recent studies on supramolecular hydrogel applications, such as cancer therapy, anti-inflammatory gels, antimicrobial activity, controlled gene drug delivery, and tissue engineering.     

ELECTROSPRAY AS AN IONISATION METHOD FOR MASS SPECTROMETRY

One of the methods by which large and non-volatile molecules, especially those of biochemical interest, can be made amenable to mass spectrometry (MS) is electrospray (ES) ionisation. In ES MS the technique of electrohydrodynamic spraying of liquids is used to produce gas-phase ions from sample molecules present in a diluted solution, which are subsequently transferred into a mass analyser. The special conditions required for effective ion formation (voltages, flow rates, solution parameters such as conductivity, sample concentration, solvent composition) are described, as well as the instrumentation used to transfer and mass-analyse the gas-phase ions. Several variants of the ES MS technique are presented which have been developed to meet certain analytical requirements (such as spraying of highly conductive or aqueous solutions, coupling to separation techniques as liquid chromatography or capillary zone electrophoresis, or the analysis of very small sample quantities). The mechanisms of liquid charging, aerosol formation and ion release from the charged droplets (as far as they are understood) are briefly discussed, and the question is addressed in how far the ions observed in the ES mass spectrum reflect the state of the sample molecules in solution. Some examples of applications from the field of peptide/protein analysis are given, covering molecular weight determination, sequence and spatial structure determination, and studies on non-covalent molecular interactions. Finally, a short comment is made on the limitations of ES MS and, from a practitoner’s point of view, which problems need still to be solved.     

Mass Spectrometric Evaluation of β-Cyclodextrins as Potential Hosts for Titanocene Dichloride

Bent metallocene dichlorides (Cp₂MCl₂, M = Ti, Mo, Nb, …) have found interest as anti-cancer drugs in order to overcome the drawbacks associated with platinum-based therapeutics. However, they suffer from poor hydrolytic stability at physiological pH. A promising approach to improve their hydrolytic stability is the formation of host-guest complexes with macrocyclic structures, such as cyclodextrins. In this work, we utilized nanoelectrospray ionization tandem mass spectrometry to probe the interaction of titanocene dichloride with β-cyclodextrin. Unlike the non-covalent binding of phenylalanine and oxaliplatin to β-cyclodextrin, the mixture of titanocene and β-cyclodextrin led to signals assigned as [βCD + Cp₂Ti–H]⁺, indicating a covalent character of the interaction. This finding is supported by titanated cyclodextrin fragment ions occurring from collisional activation. Employing di- and trimethylated β-cyclodextrins as hosts enabled the elucidation of the influence of the cyclodextrin hydroxy groups on the interaction with guest structures. Masking of the hydroxy groups was found to impair the covalent interaction and enabling the encapsulation of the guest structure within the hydrophobic cavity of the cyclodextrin. Findings are further supported by breakdown curves obtained by gas-phase dissociation of the various complexes.     

May the Best Molecule Win: Competition ESI Mass Spectrometry

Electrospray ionization mass spectrometry has become invaluable in the characterization of macromolecular biological systems such as nucleic acids and proteins. Recent advances in the field of mass spectrometry and the soft conditions characteristic of electrospray ionization allow for the investigation of non-covalent interactions among large biomolecules and ligands. Modulation of genetic processes through the use of small molecule inhibitors with the DNA minor groove is gaining attention as a potential therapeutic approach. In this review, we discuss the development of a competition method using electrospray ionization mass spectrometry to probe the interactions of multiple DNA sequences with libraries of minor groove binding molecules. Such an approach acts as a high-throughput screening method to determine important information including the stoichiometry, binding mode, cooperativity, and relative binding affinity. In addition to small molecule-DNA complexes, we highlight other applications in which competition mass spectrometry has been used. A competitive approach to simultaneously investigate complex interactions promises to be a powerful tool in the discovery of small molecule inhibitors with high specificity and for specific, important DNA sequences.     

Characterization of N-acylphosphatidylethanolamine and acylphosphatidylglycerol in oats

Two polar lipid classes, both with three acyl groups, were isolated from an extract of oats and characterized by nuclear magnetic resonance spectroscopy, electrospray mass spectrometry (MS), and electron ionization MS (EIMS). Distortionless enhancement by polarization transfer (DEPT) and the two-dimensional correlation experiments 1H-detected heteronuclear multiple quantum coherence spectroscopy, heteronuclear multiple bond correlation spectroscopy, double quantum filtered correlation spectroscopy, and total correlation spectroscopy provided sufficient information for determination of the structure of the two lipid classes. The polar lipid classes were found to be N-acylphosphatidylethanolamine [1,2-diacyl-sn-glycero-3-phospho-(N-acyl)-1'-ethanolamine; N-acyl-PE] and acylphosphatidylglycerol [1,2-diacyl-sn-glycero-3-phospho-(3'-acyl)-1'-sn-glycerol]. High-performance liquid chromatography with electrospray ionization MS (HPLC-ESMS) and with electrospray ionization tandem MS (HPLC-MS/MS) were utilized for the separation and subsequent determination of molecular species. With HPLC-ESMS, ions of deprotonated molecules were obtained and with HPLC-MS/MS carboxylate ions (representing acyl groups) were obtained as well as other structurally significant ions. Fifty molecular species of N-acylphosphatidylethanolamine and 24 molecular species of acylphosphatidylglycerol were found, with a molecular mass range of 924-1032 Da and 959-1035 Da, respectively. Identification of the fatty acid isomers, as picolinyl ester derivatives, was done with gas chromatography with EIMS. Three isomers of 16:1 fatty acids were found in N-acyl-PE, and their double bond positions were determined to 6, 9, and 11 with a relative abundance of 4:10:1.     

Generation of biologically active nano-aerosol by an electrospray-neutralization method

A simple method for manufacturing biological nano-aerosols is described. It is based on gas-phase neutralization of a cloud of highly charged electrospray-generated particles or macromolecular ions with a cloud of oppositely charged electrospray products, e.g., ions of a volatile solvent. It was demonstrated that the electrosprayed products became neutralized within a few seconds, forming a stable nano-aerosol composed of single polymer molecules, nanofibers, or nano-clusters of different sizes, depending on polymer concentration, solvent, humidity, and other factors. It was also demonstrated that enzymes aerosolized by this mild technique retain their specific activity, which opens a variety of new applications for this technology.     

Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids

Atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) has proven to be a very valuable technique for analysis of lipids from a variety of classes. This instrumental method readily produces useful ions with gentle fragmentation from large neutral molecules such as triacylglycerols and carotenoids, which are often difficult to analyze using other techniques. Molecules that are easily ionized, such as phospholipids, produce molecular ions and diagnostically useful fragment ions that are complementary to those produced by methods such as electrospray ionization MS with collision-induced dissociation. The simplicity and versatility of APCI-MS make it an ideal tool for use in solving hitherto very difficult analytical problems.     

Nanostructured materials with biomimetic recognition abilities for chemical sensing

Binding features found in biological systems can be implemented into man-made materials to design nanostructured artificial receptor matrices which are suitable, e.g., for chemical sensing applications. A range of different non-covalent interactions can be utilized based on the chemical properties of the respective analyte. One example is the formation of coordinative bonds between a polymerizable ligand (e.g., N-vinyl-2-pyrrolidone) and a metal ion (e.g., Cu(II)). Optimized molecularly imprinted sensor layers lead to selectivity factors of at least 2 compared to other bivalent ions. In the same way, H-bonds can be utilized for such sensing purposes, as shown in the case of Escherichia coli. The respective molecularly imprinted polymer leads to the selectivity factor of more than 5 between the W and B strains, respectively. Furthermore, nanoparticles with optimized Pearson hardness allow for designing sensors to detect organic thiols in air. The ‘harder’ MoS₂ yields only about 40% of the signals towards octane thiol as compared to the ‘softer’ Cu₂S. However, both materials strongly prefer molecules with -SH functionality over others, such as hydrocarbon chains. Finally, selectivity studies with wheat germ agglutinin (WGA) reveal that artificial receptors yield selectivities between WGA and bovine serum albumin that are only about a factor of 2 which is smaller than natural ligands.     

Analysis of phospholipids using electrospray ionisation tandem mass spectrometry

Phospholipids have widespread use as surfactants and emulsifying agents, and as ingredients with nutritional benefits. We describe an approach for their analysis, based on separation by reversed‐phase HPLC and identification and quantification by electrospray ionization MS and MS/MS. It enables the identification of phospholipids by assessing their polar head and fatty acid moieties, as well as their quantitative analysis.     

New Approaches to Solvent-Mediated Molecular Interactions

Recently developed integral equation methods for the evaluation of intermolecular and intramolecular interactions in a polar solvent medium are discussed and representative applications are presented. Examples include solvent modification of interionic interactions between atomic or molecular ions in water and liquid environment induced shifts in conformational equilibria for both non-polar and polar flexible molecules in water. Emphasis is placed on the features of these phenomena which can be traced specifically to the molecularity of the solvent and hence cannot be adequately reproduced by a simple continuum dielectric solvent representation.     

Modulation of Aptamer–Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer–ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer–ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs’ features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5′-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3′) and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer–ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer–ssDNA complex. The ssDNAs’ having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer–OTA interactions. The ssDNAs’ having less than 18 hydrogen bonds did not affect the aptamer–OTA affinity. The location of ssDNA’s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer–ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA’s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn’t affecting the G-qaudruplex’s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.     

Accurate Receptor-Ligand Binding Free Energies from Fast QM Conformational Chemical Space Sampling

Small molecule receptor-binding is dominated by weak, non-covalent interactions such as van-der-Waals hydrogen bonding or electrostatics. Calculating these non-covalent ligand-receptor interactions is a challenge to computational means in terms of accuracy and efficacy since the ligand may bind in a number of thermally accessible conformations. The conformational rotamer ensemble sampling tool (CREST) uses an iterative scheme to efficiently sample the conformational space and calculates energies using the semi-empirical ‘Geometry, Frequency, Noncovalent, eXtended Tight Binding’ (GFN2-xTB) method. This combined approach is applied to blind predictions of the modes and free energies of binding for a set of 10 drug molecule ligands to the cucurbit[n]urils CB[8] receptor from the recent ‘Statistical Assessment of the Modeling of Proteins and Ligands’ (SAMPL) challenge including morphine, hydromorphine, cocaine, fentanyl, and ketamine. For each system, the conformational space was sufficiently sampled for the free ligand and the ligand-receptor complexes using the quantum chemical Hamiltonian. A multitude of structures makes up the final conformer-rotamer ensemble, for which then free energies of binding are calculated. For those large and complex molecules, the results are in good agreement with experimental values with a mean error of 3 kcal/mol. The GFN2-xTB energies of binding are validated by advanced density functional theory calculations and found to be in good agreement. The efficacy of the automated QM sampling workflow allows the extension towards other complex molecular interaction scenarios.     

Structure and Physicochemical Properties of Glasses in the Li2S–LiPO3 System

The temperature–concentration dependence of the electrical conductivity of glasses in the Li₂O–LiPO₃ and Li₂S–LiPO₃ systems is investigated. With the use of the Tubandt method, it is demonstrated that the electric current in glasses of these systems is provided by migration of lithium ions. The concentration dependence of the electrical conductivity is interpreted using the obtained data on the IR absorption spectra, density, microhardness, ultrasonic velocity, etc. It is found that the electrical conductivity of glasses in the Li₂S–LiPO₃ system is more than 10³ times higher than that in pure LiPO₃. The observed increase in the electrical conductivity is explained by the formation of sulfur-containing polar structural–chemical groupings of the Li⁺[S^–PO_3/2] type, whose dissociation energy is lower than that of similar oxide polar structural fragments. This results in an increase in the number of lithium ions involved in the electricity transport due to an increase in the degree of dissociation of polar structural–chemical units.     

Validation of l-Tellurienylalanine as a Phenylalanine Isostere

Mass cytometry (MC) and imaging mass cytometry (IMC^TM) have emerged as important tools for the study of biological heterogeneity. We recently demonstrated the use of l -2-tellurienylalanine (TePhe), a mimic of phenylalanine (Phe), as an MC- and IMC-compatible protein synthesis reporter. In this work, the biochemical similarity of TePhe and its cognate analogue, Phe, are examined in the context of the RNase S complex. Isothermal titration calorimetry studies show that incorporation of TePhe preserves the interaction of S-peptide with S-protein, and the dissociation constants for the interaction of the Phe and TePhe peptides are within a factor of two. The resulting RNase S complex is catalytically active without significant alterations in the enzyme's kinetic parameters. Furthermore, circular dichroism spectroscopy does not reveal any changes to the secondary structure of TePhe-substituted RNase S. These findings provide strong evidence that TePhe functions as a Phe isostere in the context of a folded protein. It is anticipated that incorporation of TePhe into peptides or peptidomimetic scaffolds will enable facile generation of MC and IMC^TM probes.     

Structural characterization of triacylglycerols using electrospray ionization-MS<Superscript>n</Superscript> ion-trap MS

Structural analysis of mixtures of TAG requires the determination of the M.W. of each compound, the characterization of the structure(s) of the individual FA substituents, and identification of the relative disposition of the acyl groups on the glycerol backbone (regiospecificity). In this study we demonstrated that ion-trap MS in combination with electrospray ionization provides an easy and detailed characterization of the TAG structure. We showed that electrospray mass spectra are characterized by intense molecular ion adducts and that the acyl group disposition on the TAG backbone can be determined on the basis of relatively well-defined quantitative differences of fragment ions produced from MS² data. Moreover, additional spectral data can be generated by MS³ experiments on moieties containing individual acyl ion fragments. When hyphenated with chromatographic separations ion-trap electrospray MS might become a routine and powerful way to analyze TAG mixtures.     

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e⁻, 1H⁺ processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na⁺ and K⁺ adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.     

Significance of Circulating Cell-Free DNA Species in Non-Alcoholic Fatty Liver Disease

The pathogenetic mechanisms involved in the progression of non-alcoholic fatty liver disease (NAFLD) have not been completely elucidated, while the significance of circulating cell-free DNA (cf-DNA) species has been rarely evaluated in NAFLD. Herein, we assessed the serum levels of cf-DNA species in NAFLD patients and investigated their potential associations with patients’ characteristics and severity of liver disease. Forty-nine adult patients with NAFLD of any stage were included in this cohort study. Cf-DNA was isolated from patients’ sera and the levels of several distinct cf-DNA species including total cf-DNA, gene-coding cf-DNA, Alu repeat sequences, mitochondrial DNA copies and 5-methyl-2′-deoxycytidine were determined. Cirrhotic compared to non-cirrhotic patients had significantly lower serum levels of cf-DNA and RNAse P coding DNA as well as higher expression of 5-methyl-2′-deoxycytidine. After adjustment for the significant clinico-epidemiological factors, lower serum levels of cf-DNA or RNAse P were independently associated with the presence of cirrhosis. Serum levels of total and gene-coding DNA are associated with the presence of cirrhosis in NAFLD patients regardless of clinical or epidemiological parameters and may therefore be used as a screening tool for NAFLD progression.     

Acid‐Base and Organic‐Water Distribution Equilibria for Symmetrically‐Substituted P,P′‐Dialkyl Alkylenebisphosphonic Acids

Abstract

P,P′‐dialkyl alkylenebisphosphonic acids are powerful metal extraction reagents. The acid dissociation constants for a homologous series of aqueous‐insoluble P,P′‐di‐3‐(trimethylsilyl)‐propyl and aqueous‐soluble P,P′‐diethyl methylene‐, ethylene‐ and propylene‐ bisphosphonic acids were determined in a 70∶30 w/w methanol‐water solvent by potentiometric titration and ³¹P NMR spectrometry. The values obtained for the diethyl‐substituted acids were compared with those determined in water and used to assess the effect of the medium on the aqueous acid dissociation constants of the lipophilic series of P,P′‐di‐3‐(trimethylsilyl)propyl alkylenebisphosphonic acids. The dependence of the organic/aqueous distribution equilibrium on the aqueous acid concentration was also investigated using electrospray ionization mass spectrometry. The acid dissociation and organic/aqueous distribution properties of the substituted alkylenebisphosphonic acids are discussed in terms of their influence on metal ion extraction.