A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

Differences in oxidative susceptibilities between glycerophosphocholine and glycerophosphoethanolamine in dried scallop (Argopecten irradians) adductor muscle during storage: an oxidation kinetic assessment.

BACKGROUND: Phospholipids, the main lipid component in marine shellfish, mainly comprise glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). GPC and GPE in marine shellfish, especially scallop, carry n-3 long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), although different types of glycerophospholipids (GP) have different health benefits on human health. Moreover, different GP subclasses such as GPC and GPE have different oxidative susceptibilities in complex food systems. The present study compared the oxidative susceptibilities of GPC and GPE in dried scallop during storage by high-performance liquid chromatography-tandem mass spectrometry and kinetic models, and also investigated the effects of natural phenolic antioxidant on their susceptibilities. RESULTS: The results showed that GPC and GPE molecular species (carrying EPA or DHA) contents in samples continuously reduced during storage at two different temperatures. The first-order kinetic model better reflected the changes of GPC and GPE molecular species (carrying EPA or DHA) in samples than the zero-order kinetic model during storage. According to the oxidation rate (k) obtained from first-order kinetic models, GPE possessed a greater oxidation rate than GPC during storage. Moreover, the results showed that antioxidants of bamboo leaves (AOB, polar polyphenolic antioxidants) significantly decreased the oxidation rates of GPC and GPE molecular species (carrying EPA or DHA) in samples during storage, and GPC could be more effectively protected by AOB compared to GPE. CONCLUSION: The present study provides a practical method for accurately evaluating the oxidative susceptibility of different phospholipid classes in complex food systems. © 2020 Society of Chemical Industry.

Regiochemical Assignment of N-Acylphosphatidylethanolamines (NAPE) by Liquid Chromatography/Electrospray Ionization with Multistage Mass Spectrometry and Its Application to Extracts of Lupin Seeds.

1,2-Diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) are low abundance phospholipids but important constituents of intracellular membranes of plant tissues, responsible for generating bioactive N-acylethanolamine (NAE), which participates in several physiological processes such as regulation of seed germination and protection against pathogenic attacks. From an analytical point of view, the critical aspect of these bioactive lipids lies in the determination of fatty acyl chains located in sn-1/sn-2 position on the glycerol backbone (O-linked), along with the amide-bound (N-linked) fatty acyl chain. Here, the identity and occurrence of NAPE in lipid extracts of lupin seeds (Lupinus luteus L.) was assessed by electrospray ionization in negative ion mode upon reversed-phase liquid chromatography (RPLC-ESI) coupled to mass spectrometry (MS) either at high- (i.e., Orbitrap FTMS) or low- (linear ion trap, LIT) resolution/accuracy. Collisional induced dissociation (CID)-tandem MS and MS3 acquisitions of chemically prepared NAPE allowed to unequivocally recognize the N-linked fatty acyl chain and to establish the diagnostic product ions that were successfully applied to identify NAPE in lipid extracts of yellow lupin seeds. The most abundant NAPE species were those containing N-acyl groups C18:1, C18:2; a minor prevalence was found for C16:0, C18:0, and C18:3, and almost the same acyl chains O-linked on the glycerol backbone in several sn-1/sn-2 combinations were observed. The positional isomers of NAPE species were identified as deprotonated molecules ([M-H]-) at m/z 978.7541 (three isomers 52:3), m/z 980.7694 (two isomers 52:2), m/z 1002.7535 (four isomers 54:5), m/z 1004.7686 (two isomers 54:4), m/z 1006.7837 (two isomers 54:3), and m/z 1008.8026 (single isomer 54:2). The total amount of NAPE in lupin seeds ranged in the interval of 2.00 ± 0.13 mg/g dw, in agreement with other edible legumes. We anticipate our approach to be a robust assessment method potentially applicable to biological extracts containing NAPE species and can provide comprehensive profiles and contents.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes.

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 µm is found to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants.

Uptake and passage of nanocarriers through the placenta are critical information to develop new therapeutic approaches during pregnancy. In order to assess nanocarriers transplacental passage and penetration into the placenta, we studied and optimized two ex-vivo human models: the dually perfused placenta and the placenta explants. Doubly labelled PEGylated liposomes were used as models to provide data on the penetration and transplacental passage of drugs and liposomes. A HPLC method was set-up to quantify both carboxyfluorescein and lipid-rhodamine. Transplacental passage was then quantified using HPLC and placental penetration was assessed using spinning disk microscopy. We found a similar transplacental passage rate for both free and encapsulated carboxyfluorescein as well as a homogeneous fluorescence intensity in the outer cell layer of the placental villous, the syncytiotrophoblast, and the mesenchyma. Besides, liposome-rhodamine was not detected in the fetal circulation. The absence of transplacental passage of PEGylated liposomes is also supported by their detection in the sole syncytiotrophoblast. The combination of two ex-vivo models and the monitoring of both the drug and the carrier provided consistent and complementary information. Overall, we suggest combining the perfused human placenta and the human explants villous models to evaluate nanocarriers designed for treatments during pregnancy.

MALDI-MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance.

Mass spectrometry (MS) has been widely used for enzyme activity assays. Herein, we propose a MALDI-MS patterning strategy for the convenient visual presentation of multiple enzyme activities with an easy-to-prepare chip. The array-based caspase-activity patterned chip (Casp-PC) is fabricated by hydrophobically assembling different phospholipid-tagged peptide substrates on a modified ITO slide. The advantages of amphipathic phospholipids lead to high-quality mass spectra for imaging analysis. Upon the respective cleavage of these substrates by different caspases, such as caspase-1, -2, -3, and -8, to produce a mass shift, the enzyme activities can be directly evaluated by MALDI-MS patterning by m/z-dependent imaging of the cleavage products. The ability to identify drug-sensitive/resistant cancer cells and assess the curative effects of anticancer drugs is demonstrated, indicating the applicability of the method and the designed chip.

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry.

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery-release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.

Growth and spectral assessment of Yb(3+)-doped KBaGd(MoO4)3 crystal: a candidate for ultrashort pulse and tunable lasers.

In order to explore new more powerful ultrashort pulse laser and tunable laser for diode-pumping, this paper reports the growth and spectral assessment of Yb(3+)-doped KBaGd(MoO(4))(3) crystal. An Yb(3+):KBaGd(MoO(4))(3) crystal with dimensions of 50×40×9 mm(3) was grown by the TSSG method from the K(2)Mo(2)O(7) flux. The investigated spectral properties indicated that Yb(3+):KBaGd(MoO(4))(3) crystal exhibits broad absorption and emission bands, except the large emission and gain cross-sections. This feature of the broad absorption and emission bands is not only suitable for the diode pumping, but also for the production of ultrashort pulses and tunability. Therefore, Yb(3+):KBaGd(MoO(4))(3) crystal can be regarded as a candidate for the ultrashort pulse and tunable lasers.

Structural elucidation of molecular species of pacific oyster ether amino phospholipids by normal-phase liquid chromatography/negative-ion electrospray ionization and quadrupole/multiple-stage linear ion-trap mass spectrometry.

Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.

Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures.

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

Elastic deformations in hexagonal phases studied by small-angle X-ray diffraction and simulations.

In this study we present experimental and theoretical results which concern the deviations from circularity of the pivotal plane in the inverse hexagonal phases (H(II)) of phospholipid self-assemblies. Due to packing constraints, the cross-section of the polar/apolar interface deviates from a circle, which we studied in minute detail by analysing small-angle X-ray diffraction data of dioleoyl-phosphatidylethanolamine (DOPE) and stearoyl-oleoyl-phosphatidylethanolamine (SOPE), respectively. On this structural basis, Monte Carlo (MC) simulated annealing variations of the free energy were carried out, both on the formation of the H(II)-phase and on the particular shape of the cross-section in the H(II)-phase. The equilibrium of the H(II)-phase pivotal plane contour and the corresponding values of the mean intrinsic curvature, H(m), and the hydrocarbon chain stiffness, τ, were determined from MC calculations. The results of these calculations were tested by solving the corresponding system of non-linear differential equations derived using variational calculus. Here our main aim is to predict the range of possible values of H(m) and τ. Comparing the measured structural data with predictions from MC calculations including lipid anisotropy, and accounting for the elastic deformations of the pivotal plane allowed us to determine a relationship between the bending deformation and stretching of hydrocarbon chains.

Persistent voids: a new structural metric for membrane fusion.

MOTIVATION: Membrane fusion constitutes a key stage in cellular processes such as synaptic neurotransmission and infection by enveloped viruses. Current experimental assays for fusion have thus far been unable to resolve early fusion events in fine structural detail. We have previously used molecular dynamics simulations to develop mechanistic models of fusion by small lipid vesicles. Here, we introduce a novel structural measurement of vesicle topology and fusion geometry: persistent voids. RESULTS: Persistent voids calculations enable systematic measurement of structural changes in vesicle fusion by assessing fusion stalk widths. They also constitute a generally applicable technique for assessing lipid topological change. We use persistent voids to compute dynamic relationships between hemifusion neck widening and formation of a full fusion pore in our simulation data. We predict that a tightly coordinated process of hemifusion neck expansion and pore formation is responsible for the rapid vesicle fusion mechanism, while isolated enlargement of the hemifusion diaphragm leads to the formation of a metastable hemifused intermediate. These findings suggest that rapid fusion between small vesicles proceeds via a small hemifusion diaphragm rather than a fully expanded one. AVAILABILITY: Software available upon request pending public release. SUPPLEMENTARY INFORMATION: Supplementary data are available on Bioinformatics online.

Assess the nature of cholesterol-lipid interactions through the chemical potential of cholesterol in phosphatidylcholine bilayers.

Cholesterol plays a vital role in determining the physiochemical properties of cell membranes. However, the detailed nature of cholesterol-lipid interactions is a subject of ongoing debate. Existing conceptual models, including the Condensed Complex Model, the Superlattice Model, and the Umbrella Model, identify different molecular mechanisms as the key to cholesterol-lipid interactions. In this work, the compositional dependence of the chemical potential of cholesterol in cholesterol/phosphatidylcholine mixtures was systematically measured at high resolution at 37 degrees C by using an improved cholesterol oxidase (COD) activity assay. The chemical potential of cholesterol was found to be much higher in di18:1-PC bilayers than in di16:0-PC bilayers, indicating a more favorable interaction between cholesterol and saturated chains. More significantly, in 16:0,18:1-PC and di18:1-PC bilayers, the COD initial-reaction rate displays a series of distinct jumps near the cholesterol mole fractions (chi(C)) of 0.15, 0.25, 0.40, 0.50, and 0.57 and a peak at the cholesterol maximum solubility limit of 0.67. These jumps have been identified as the thermodynamic signatures of stable cholesterol regular distributions. In contrast, no such jumps were evident in di16:0-PC bilayers below chi(C) of 0.57. The observed chemical potential profile is in excellent agreement with previous Monte Carlo simulations based on the Umbrella Model but not with the predictions from the other models. The data further indicate that the cholesterol regular distribution domains (superlattices) are not the hypothesized condensed complexes. Those complexes were mainly implicated from studies on lipid monolayer that may not be relevant to the lipid bilayer in cell membranes.

Origin of short-range repulsion between hydrated phospholipid bilayers: a computer simulation study.

The grand canonical Monte Carlo technique is used to simulate the pressure-distance dependence for supported dilauroylphosphatidylethanolamine (DLPE) membranes. The intra- and intermolecular interactions in the system are described with a combination of an AMBER-based force field for DLPE and a TIP4P model for water. To improve the balance between the pair interactions of like and unlike molecules, the water-lipid interaction potentials are scaled to reproduce the hydration level and intermembrane separation at full hydration. It is found that the short-range water-mediated repulsion originates from the hydration component of the intermembrane pressure, whereas the direct interaction between the membranes remains attractive throughout the pressure range studied (0-5 kbar).

Direct computer simulation of water-mediated force between supported phospholipid membranes.

The grand canonical Monte Carlo technique is used to calculate the water-mediated force operating between two supported 1,2-dilauroyl-DL-phosphatidylethanolamine (DLPE) membranes in the short separation range. The intra- and intermolecular interactions in the system are described with a combination of an AMBER-based force field for DLPE and a TIP4P model for water. The long range contributions to the electrostatic interaction energy are treated in the dipole-dipole group-based approximation. The total water-mediated force is analyzed in terms of its hydration component and the component due to the direct interaction between the membranes. The latter is, in addition, partitioned into the electrostatic, van der Waals, and steric repulsion contributions to give an idea of their relative significance in the water-mediated interaction of the membranes.

Force barriers for membrane tube formation.

We used optical tweezers to measure the force-extension curve for the formation of tubes from giant vesicles. We show that a significant force barrier exists for the formation of tubes, which increases linearly with the radius of the area on which the pulling force is exerted. The tubes form through a first-order transition with accompanying hysteresis. We confirm these results with Monte Carlo simulations and theoretical calculations. Whether membrane tubes can be formed in, for example, biological cells, thus depends on the details of how forces are applied.

Experimental and Monte Carlo simulation studies of the thermodynamics of polyethyleneglycol chains grafted to lipid bilayers.

Experimental measurements of the affinity of binding of fluorescent acylated polyethyleneglycol (PEG) conjugates to bilayers containing varying levels of phosphatidylethanolamine-PEGs (PE-PEGs) have been combined with Monte Carlo simulations to investigate the properties of the polymer chains at a PEG-grafted lipid interface. The affinity of binding of such conjugates to large unilamellar phosphatidylcholine/phosphatidylethanolamine (9:1) vesicles decreases 27-fold as the size of the coupled PEG chain increases from 1 to 114 monomer units. Incorporation of increasing amounts of PE-PEG2000 or PE-PEG5000 into the vesicles progressively reduces the affinity of binding of acylpeptide-PEG2000 or -PEG5000 conjugates. Monte Carlo simulations of surfaces with grafted PEG chains revealed no significant dependence of several characteristic properties of the polymer chains, including the average internal energy per polymer and the radii of gyration, on the grafting density in the range examined experimentally. The average conformation of a surface-grafted PEG2000 or PEG5000 chain was calculated to be fairly extended even at low grafting densities, and the projected cross-sectional areas of the grafted PEG chains are considerably smaller than those predicted on the basis of the estimated Flory radius. The experimental variation of the binding affinity of acylated conjugates for bilayers containing varying mole fractions of PE-PEG2000 or -PEG5000 is well explained by expressions treating the surface-grafted PEG polymers either as a van der Waals gas or as a system of rigid discs described by scaled particle theory. From the combined results of our experimental and simulation studies we conclude that the grafted PEG chains exist in a "mushroom" regime throughout the range of polymer densities examined experimentally and that the diminished affinity of binding of acylated-PEG conjugates to bilayers containing PE-PEGs results from occlusion of the surface area accessible for conjugate binding by the mobile PE-PEG polymer chains.

Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.

This paper describes the use of positive and negative ion electrospray mass spectrometry (MS) and MS/MS (tandem mass spectrometry) to identify glycerophospholipid and ceramide headgroups and their alkyl, alkenyl and acyl constituents. Molecular ion adducts were the primary products formed by positive ionization, occurring as [M+H]+, [M+Na]+, [M+K]+, [M+formate]+, or [M+acetate]+, depending upon the class of glycerophospholipid and the presence or absence of these ionization-promoting species. Similar (negatively charged) ions corresponding to the loss of the groups listed above were formed in negative ion MS. Positive ion electrospray MS/MS provides information on the nature of the headgroup, with the formation of an ion corresponding to the headgroup itself, or the loss of the headgroup from the molecular ion H+ or Na+ adduct. Acyl constituents are identified during negative ion MS/MS from the formation of their RCOO- ions. The nature of alkyl or alkenyl substituents in glycerophosphoethanolamine (PE) molecular species can be identified from residual ions following the loss of ethanolamine plus loss of the acyl moiety in the sn-2 position, and cyclization of a phosphate oxygen with C-2 of glycerol. In glycerophosphoinositol (PI) species, it appears that an RCO- ion is formed during negative ion MS/MS, possibly to steric interference from the bulky phosphoinositol headgroup that prevents cyclization (and subsequent stabilization) of the ion described for PE species. Positive and negative ion electrospray MS spectra for molecular species of commercial preparations of PE, PI, phosphatidylserine (PS), glycerophosphocholine (PC) and sphingomyelin (SM) produced similar profiles. For phospholipids occurring as Na+ adducts, concentrations above ca. 1 ng/microliter produced significant quantities of both [M+H]+ and [M+Na]+ ions for those molecular species present in the largest quantities, complicating interpretation of the spectra. Complete profiles of molecular species were obtained from as little as 10 picograms of material. Major components of PE were identified from 0.1 picogram total lipid. Using single ion monitoring of the Na+ adduct of beta-acetyl-gamma-O-hexadecyl L-alpha-phosphatidylcholine, 10 femtograms of material was detected. A mixture of 1 nanogram each of PE, PI, PS, and PC was readily resolved into individual molecular species, with little apparent loss of resolution or preferential ionization. Electrospray MS did not provide information on the position (sn-1 or sn-2) of fatty acids, and was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without prior separation of these lipid subclasses.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of protein binding to receptor-doped lipid monolayers by Monte Carlo simulation.

This paper presents a Monte Carlo simulation (MCS) method for estimating the parameters that characterize ligand-receptor binding directly from experimentally derived binding isotherms. Binding parameters are estimated by incorporating an MCS algorithm for ligand binding to a two-dimensional receptor array into a nonlinear regression program. The MCS method was tested by analyzing experimental isotherms of avidin binding to biotinylated lipid in Langmuir-Blodgett (LB) monolayers. The MCS-derived cooperativity coefficients and intrinsic association constants for avidin-biotin binding to LB films are correlated strongly (R2 > 0.93) with the binding parameters determined from the same experimental data by a thermodynamic equilibrium binding model (Zhao et al. 1993. Langmuir. 9:3166-3173). This result shows MCS to be an accurate and potentially more versatile method for characterizing biomolecular interactions at surfaces.