Characterization of sodiated glycerol phosphatidylcholine phospholipids by mass spectrometry

Fast atom bombardment mass spectrometry in the positive mode was used for the characterization of sodiated glycerol phosphatidylcholines. The relative abundance (RA) of the protonated species is similar to the RA of the sodiated molecular species. The sodiated fragment ion, [M + Na - 59](+), corresponding to the loss of trimethylamine, and other sodiated fragment ions, were also observed. The decomposition of the sodiated molecule is very similar for all the studied glycerol phosphatidylcholines, in which the most abundant ion corresponds to a neutral loss of 59 Da. Upon collision-induced dissociation (CID) of the [M + Na](+) ion informative ions are formed by the losses of the fatty acids in the sn-1 and sn-2 positions. Other major fragment ions of the sodiated molecule result from loss of non-sodiated and sodiated choline phosphate, [M + Na - 183](+), [M + Na - 184](+.) and [M + Na - 205](+), respectively. The main CID fragmentation pathway of the [M + Na - 59](+) ion yields the [M + Na - 183](+) ion, also observed in the CID spectra of the [M + Na](+) molecular ion. Other major fragment ions are [M + Na - 205](+) and the fragment ion at m/z 147. Collisional activation of [M + Na - 205](+) results in charge site remote fragmentation of both fatty acid alkyl chains. The terminal ions of these series of charge remote fragmentations result from loss of part of the R(1) or R(2) alkyl chain. Other major informative ions correspond to acylium ions.     

Rapid and selective identification of molecular species in phosphatidylcholine and sphingomyelin by conditional neutral loss scanning and MS3

Analyses of molecular species of phospholipids containing choline (Ch), such as phosphatidylcholine (PC) and sphingomyelin (SM), are reported. Neutral loss scanning was applied for the selective detection of these lipids using a quadrupole-linear ion trap mass spectrometer. By using ammonium formate as an elution buffer, both PC and SM were detected as [M+HCOO]- ions in the negative ion mode. Upon collisional activation, the [M+HCOO]- adduct ions underwent facile elimination of HCO2, to yield an ion which, in turn, readily underwent collisional-induced dissociation (CID) to eliminate CH3 to yield an [M-CH3]- ion. By selecting the proper conditions for scanning for neutral loss of 60 Da (HCO2+CH3), SM species were identified separately from PCs. Further, by selection of this [M-CH3]- ion as the precursor ion, the identities of the fatty acyl chains of PC species can be effectively obtained by MS3 experiments. Furthermore, by the MS3 analyses of [M-CH3]- specifically obtained from SM molecules, identification of sphingosine or sphinganine derivatives and their N-acyl species can also be effectively obtained. This systematic analysis of PCs and SMs by conditional neutral loss scanning, with subsequent analyses by MS3, using a linear ion trap mass spectrometer in the negative ion mode, appears to be a very effective and sensitive method. Further, MS/MS in the positive ion mode at relatively low collision energy was also effective for the identification of positional specificities in individual molecular PC species from their lysoPC-related fragments. The present paper deals only with qualitative identification of individual molecular species, and the related quantitative studies are now underway.     

Determination of phosphorus:glycerol:acyl ratios in phospholipids

Phospholipids (1–5 mg) are mixed with methyl heptadecanoate as an internal standard. One part of the sample is reduced with Vitride in tetrahydrofuran in a sealed tube at 50 2C for 1 hr. The reaction products are acetylated in the same tube by treatment with acetic acid-acetic anhydride at 140 °C. After 1 hr total O-acyl and after 16 additional hours glycerol are determined as the fully acetylated compounds by gas-liquid chromatography.Another part of the sample is subjected to acidic hydrolysis and total O-acyl and N-acyl groups are determined by GLC as fatty-acid methyl esters. The aqueous phase is heated to 100 °C with 6 N HCl for 72 hr and phosphorus is measured colorimetrically.     

Vicinal hydroxylation of unsaturated fatty acids for structural characterization of intact neutral phospholipids by negative electrospray ionization tandem quadrupole mass spectrometry

We report a novel method allowing the complete structural characterization of intact species of the phospholipid classes phosphatidylcholine and phosphatidylethanolamine by utilizing negative electrospray ionization quadrupole tandem mass spectrometry (MS/MS). Information on the molecular weight of the intact phospholipid species, the class to which it belongs, the molecular mass of the fatty acid substituents and their regioisomerism, is easily revealed by MS/MS. Throughout our investigations the R2COO- ions were more abundant than the R1COO- ions, and this observation is used for regioisomeric assignment of the two fatty acids. However, for phospholipid species containing an unsaturated fatty acid, information on the position of the double bond is not achieved in this way. By converting the olefinic sites to their 1,2-dihydroxylated derivatives, information on the position of the hydroxyl groups (and hence of the double bond) is obtained by performing a second MS/MS experiment. Thus, a complete structural characterization of intact phosphatidylcholine and phosphatidylethanolamine species is obtained by performing these two MS/MS experiments. In order to ensure structural distinction of isobaric species, a number of phosphatidylethanolamine and phosphatidylcholine species were synthesized from lyso-phosphatidylcholine and analyzed by the present method. The applicability of the method to real samples is also demonstrated by the complete structural elucidation of the two phosphatidylcholine species 16:0/18:1Delta9 and 16:0/18:1Delta11 from egg yolk.     

Evaluation of urinary ribonucleoside profiling for clinical biomarker discovery using constant neutral loss scanning liquid chromatography/tandem mass spectrometry

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.     

Electron energy loss spectroscopy of liquid glycerol

The inelastic scattering of low energy electrons from liquid glycerol has been studied. For the first time, electron energy loss spectra of liquids are sufficiently well resolved to permit the identification of vibrations corresponding to individual bonds, namely the C–H and O–H stretching vibrations in glycerol. The angular distribution of the specular peak is very broad, indicating the absence of long-range order at the surface of the liquid. The measurement of the loss signals as a function of the primary electron energy suggests a hybrid mechanism of excitation. The excitation mechanism for the O–H vibration has a stronger impact character as compared to the C–H vibration. A negative ion resonance of glycerol is found at a primary energy of 8 eV. The signal intensities measured as a function of the specular angle of the electron beam appear to be influenced by the angular dependence of the dipole and impact scattering cross-section and a possible preferred orientation of the C–H and O–H groups at the surface of the liquid.     

Selective detection of nitrated polycyclic aromatic hydrocarbons by electrospray ionization mass spectrometry and constant neutral loss scanning

The development of a method for selective detection of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) among other polycyclic aromatic compounds (PACs) is described. The method is based on electrospray ionization mass spectrometry (ESI-MS), performed with a triple quadrupole analyzer and constant neutral loss (CNL) scanning. When subjected to ESI conditions, nitro-PAHs give rise to M(-), [M - H](-) and [M - H + 16](-) ions, which in turn produce fragments by losing 30 u, most probably NO. Other PACs do not undergo such fragmentations, and these differences can be exploited for selective detection of nitro-PAHs among other PACs. Nitro-PAHs can therefore be monitored through the loss of 30 u occurring under negative ion mode ESI conditions. Toward the full development of a screening method for nitro-PAHs, this article first discusses some general aspects of the negative ion mode full-scan ESI mass spectra obtained for these compounds and other PAH derivatives. Because the extent of observation of the loss of 30 u is sensitive to the ESI conditions used, the effects of ionization parameters such as solvent used, declustering voltage, and solvent flow rate are evaluated and discussed. Setting these parameters is very important, especially when interfacing a high performance liquid chromatography (HPLC) system with the ESI source of a triple quadrupole mass spectrometer. Preliminary results of on-line microbore HPLC/ESI-MS separations of PAC standards are presented, and elution/ionization conditions discussed.     

Synthesis and characterization of carbohydrate-based phospholipids

Novel carbohydrate-based phospholipids containing two saturated C(12) (dilauroyl ribo-phosphocholine) (DLRPC), C(14) (dimyristoyl ribo-phosphocholine) (DMRPC), and C(20) (diarachadonyl ribo-phosphocholine) (DARPC) carboxylic acid chains were synthesized. The physical properties of the supramolecular structures formed by these compounds were compared to those formed by their direct glycerol analogues dilauroyl phosphocholine (DLPC), dimyristoyl phosphocholine (DMPC), and diarachadonyl phosphocholine (DAPC). Modulated differential scanning calorimetry (MDSC) and X-ray diffraction data indicated that with chain lengths < or =14 carbons, the carbohydrate backbone increased the thermal stability of the bilayer below the phase-transition temperature (T(m)) as compared to the glycerol-based lipids. With longer chains (C(20)), the bilayer structure was destabilized as compared to glycerol-based lipids. NMR studies of a DMRPC vesicle dispersion reveal split choline headgroup signals and distinct magnetization transfer effects arising from the "inner" and "outer" surfaces of the bilayer vesicle. Modulated differential scanning calorimetry also demonstrated that glycerol- and carbohydrate-based lipids mix, as evidenced by a single intermediate T(m). In addition, carbohydrate-based lipid/cholesterol mixtures exhibited a decrease in enthalpy with an increase in cholesterol concentration. Unlike glycerol phospholipids, carbohydrate lipids were resistant to enzymatic degradation by phospholipase A(2) (PLA(2)).     

Constant loss in Brillouin spectra of polymers

The polarized (VV) and depolarized (VH) light scattering spectra of polyisobutylene, poly(methyl methacrylate), and glycerol were measured in the gigahertz frequency range at temperatures below and above the glass transition. Both VV and VH spectra exhibit a significant constant loss contribution that appears as a frequency‐independent imaginary part of the susceptibility spectrum. Existence of the frequency‐independent susceptibility in VV spectra below the Brillouin lines suggests that the constant loss also appears in mechanical relaxation in the gigahertz frequency range. Intensity of the constant loss increases strongly with temperature. Analysis of the spectra and literature data suggests that the constant loss can be general for many glass‐forming systems, but it is hidden in many cases by other relaxation contributions. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 40: 201–209, 2002     

High-performance liquid chromatographic separation of molecular species of neutral phospholipids.

Molecular species of neutral phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were resolved by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases of acetonitrile-methanol-water containing tetraalkylammonium phosphates (TAAPs). Competitive interactions of TAAPs and analyte solutes with a reversed-phase HPLC column resulted in reduced retention of PC or PE with concomitant increase in detection sensitivity. The chromatographic data for PC and PE were distinctly different from those for negatively charged phospholipids where ion-pair retention mechanisms prevailed. While PC (or PE) components eluted at longer retention times with a larger size of TAAP, an increase in the TAAP concentration invariably caused a decrease in phospholipid retention times. Optimization of HPLC conditions by using high concentrations (25-100 mM) of tetramethylammonium phosphate in acetonitrile-methanol-water (70:22:8) facilitated elution of components with improved peak symmetry. HPLC separations of neutral phospholipids derived from animal sources were more complex than those from soybeans.     

Reference scales for the characterization of cationic electrophiles and neutral nucleophiles

Twenty-three diarylcarbenium ions and 38 pi-systems (arenes, alkenes, allyl silanes and stannanes, silyl enol ethers, silyl ketene acetals, and enamines) have been defined as basis sets for establishing general reactivity scales for electrophiles and nucleophiles. The rate constants of 209 combinations of these benzhydrylium ions and pi-nucleophiles, 85 of which are first presented in this article, have been subjected to a correlation analysis to determine the electrophilicity parameters E and the nucleophilicity parameters N and s as defined by the equation log k(20 degrees C) = s(N + E) (Mayr, H.; Patz, M. Angew. Chem., Int. Ed. Engl. 1994, 33, 938-957). Though the reactivity scales thus obtained cover more than 16 orders of magnitude, the individual rate constants are reproduced with a standard deviation of a factor of 1.19 (Table 1). It is shown that the reactivity parameters thus derived from the reactions of diarylcarbenium ions with pi-nucleophiles (Figure 3) are also suitable for characterizing the nucleophilic reactivities of alkynes, metal-pi-complexes, and hydride donors (Table 2) and for characterizing the electrophilic reactivities of heterosubstituted and metal-coordinated carbenium ions (Table 3). The reactivity parameters in Figure 3 are, therefore, recommended for the characterization of any new electrophiles and nucleophiles in the reactivity range covered. The linear correlation between the electrophilicity parameters E of benzhydryl cations and the corresponding substituent constants sigma(+) provides Hammett sigma(+) constants for 10 substituents from -1.19 to -2.11, i.e., in a range with only very few previous entries.     

On the low frequency loss peak in the dielectric spectrum of glycerol

We measured dielectric spectra of glycerol at pressures exceeding 1 GPa in order to examine the slow Debye-like peak. This peak is not a relaxation process, but its frequency is consistent with an origin in dielectric discontinuities due to impurities. These heterogeneities have a non-negligible bulk modulus and are identified as volatile, relatively non-polar liquid contaminants. Although this slow peak is often found in the dielectric spectra of polyalcohols, it is not an intrinsic feature thereof, unlike the ostensibly similar relaxation peak in monoalcohols.     

Application of scanning electrochemical microscopy and scanning electron microscopy for the characterization of carbon-spray modified electrodes

Different gold surfaces modified by carbon-spray have been investigated by scanning electron microscopy (SEM) and scanning electrochemical microscopy (SECM). A transformation of the SECM image to a distance-location profile is proposed which assists the correlation of both images. The structures found in the transformed SECM images of carbon-spray layers on gold substrates can be explained by the topographic features visible in the SEM pictures. Tempering the carbon spray results in an increased density of electrochemically reactive carbon particles which could be confirmed by cyclic voltammetric investigations. Gold minigrids modified with carbon spray expose some areas of especially large currents which could not be predicted from their SEM images. This effect may result from particles located at the edge of a wire intersection having relatively large active surfaces per particle. They contribute significantly to the total current of the minigrid.     

Scaling of the hysteresis in the glass transition of glycerol with the temperature scanning rate

By measuring the dependences of the temperature-dependent primary ("alpha") dielectric relaxation time behavior on the temperature scanning rate for the glass-forming glycerol, we study the scaling of hysteresis at the glass transition in glycerol. Based on the Vogel-Fulcher-Tammann (VFT) expression and the Angell's fragility concept, notable correlations of the systematic kinetic fragility, and of the hysteresis effect in the vitrification∕fusion "alpha"-relaxation process of glycerol, with the temperature scanning rate, were reasonably analyzed and discussed. It was observed that the kinetic fragility m and the apparent glass-transition temperature hysteresis width ΔT(g)(a), respectively, scaled the temperature scanning rate q as m ≈ α(m)q(-γ) and ΔT(g)(a) ≈ A(0) + αq(β), at which the exponents, γ and β, were suggested to be characteristic of the resistance to the structure change or fragility change of the system during the glass transition. The observed scaling laws are quite similar to the scaling power law for the thermal hysteresis in the first-order phase transition (FOPT) of solids, providing a significant insight into the hysteresis effect in the glass transition of the glass-forming liquids.     

Iodoacetamide-alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano-electrospray ionization quadrupole time-of-flight parent ion scanning

Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS = 105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da +22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed.     

Detailed structure of hairy mixed micelles formed by phosphatidylcholine and PEGylated phospholipids in aqueous media

Aqueous dispersions of mixed egg yolk phosphatidylcholine (PC) and poly(ethylene glycol) (PEG) modified distearoyl phosphatidylethanolamine (DSPE) were investigated with the purpose of determining shape, size, and conformation of the formed mixed micelles. The samples were prepared at a range of DSPEPEG to PC molar ratios ([DSPEPEG/PC] from 100:0 to 30:70) and with, respectively, DSPEPEG2000 and DSPEPEG5000, where 2000 and 5000 refer to the molar masses of the PEG chains. Particle shape and internal structure were studied using small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The contrast of the micelles is different for X-rays and neutrons, and by combining SANS and SAXS, complementary information about the micelle structure was obtained. The detailed structure of the micelles was determined in a self-consistent way by fitting a model for the micelles to SANS and SAXS data simultaneously. In general, a model for the micelles with a hydrophobic core, surrounded by a dense hydrophilic layer that is again surrounded by a corona of PEG chains in the form of Gaussian random coils attached to the outer surface, is in good agreement with the scattering data. At high DSPEPEG contents, nearly spherical micelles are formed. As the PC content increases the micelles elongate, and at a DSPEPEG/PC ratio of 30:70, rodlike micelles longer than 1000 angstroms are formed. We demonstrate that by mixing DSPEPEG and PC a considerable latitude in controlling the particle shape is obtained. Our results indicate that the PEG chains in the corona are in a relatively unperturbed Gaussian random coil conformation even though the chains are far above the coil-coil overlap concentration and, therefore, interpenetrating. This observation in combination with the observed growth behavior questions that the "mushroom-brush"transition is the single dominating factor for determining the particle shape as assumed in previous theoretical work (Hristova, K.; Needham, D. Macromolecules 1995, 28, 991-1002).     

The role of glycerol and phosphatidylcholine in solubilizing and enhancing insulin stability in reverse hexagonal mesophases

The potential of reverse hexagonal mesophases based on monoolein (GMO) and glycerol (as cosolvent) to facilitate the solubilization of proteins, such as insulin was explored. H(II) mesophases composed of GMO/decane/water were compared to GMO/decane/glycerol/water and GMO/phosphatidylcholine (PC)/decane/glycerol/water systems. The stability of insulin was tested, applying external physical modifications such as low pH and heat treatment (up to 70°C), in which insulin is known to form ordered amyloid-like aggregates (that are associated with several neurodegenerative diseases) with a characteristic cross β-pleated sheet structure. The impact of insulin confinement within these carriers on its stability, unfolding, and aggregation pathways was studied by combining SAXS, FTIR, and AFM techniques. These techniques provided a better insight into the molecular level of the "component interplay" in solubilizing and stabilizing insulin and its conformational modifications that dictate its final aggregate morphology. PC enlarged the water channels while glycerol shrank them, yet both facilitated insulin solubilization within the channels. The presence of glycerol within the mesophase water channels led to the formation of stronger hydrogen bonds with the hosting medium that enhanced the thermal stability of the protein and remarkably affected the unfolding process even after heat treatment (at 70°C for 60 min).     

Differential scanning calorimetry: A powerful tool for the characterization of thermoplastics

The use of differential scanning calorimetry for examining the thermal behaviour of thermoplastics is described. Applications which are discussed include the measurement of specific heat, glass transition phenomena, melting and crystallization characteristics and thermal stability. The value of the results is demonstrated by means of a few examples in each area of application.