Mass spectrometry of the phosphatidylcholines: fragmentation processes for dioleoyl and stearoyl-oleoyl glycerylphosphorylcholine

Mass spectra for the various phosphatidylcholines, together with accurate mass measurements on the more abundant fragment ions, have been described in a previous paper (Ref. 5). No detailed fragmentation sequence was proposed on the evidence available. In the case of dioleoyl glycerylphosphorylcholine, some question arose as to whether certain ions were produced by electron impact or by pyrolysis. In this paper, results are reported which enable a more detailed fragmentation sequence to be proposed. By observing metastable transitions in the first field free region of a double-focusing mass spectrometer, it can be shown that the major ions in the spectrum are produced by electron impact processes, and not by pyrolysis; moreover, many of these ions are directly related to one another by metastable processes. In particular, it has been demonstrated that the ions at m/e 603 for dioleoyl glycerylphosphorylcholine and at m/e 604 for stearoyl-oleoyl glycerylphosphorylcholine are derived from the appropriate molecular ions by an electron impact-induced process. From measurements of the metastable ion intensities, as well as from the appearance potentials and ionization efficiency curves, conclusions may be drawn about many of the fragmentation mechanisms, allowing a distinction to be made between rearrangement and cleavage reactions.     

Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation

The molecular composition of phosphatidylcholines (PCs) in total lipid extracts was characterized by a combination of multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer and MS3 fragmentation on an ion trap mass spectrometer. Precursor ion spectra for 50 acyl anion fragments of fatty acids (fatty acid scanning) acquired in parallel increased the specificity and the dynamic range of the detection of PCs and identified the fatty acid moieties in individual PC species. Subsequent analysis of detected PC peaks by MS3 fragmentation on an ion trap mass spectrometer quantified the relative amount of their positional isomers, thus providing the most detailed and comprehensive characterization of the molecular composition of the pool of PCs at the low-picomole level. The method is vastly simplified, compared with conventional approaches, and does not require preliminary separation of lipid classes or of individual molecular species, enzymatic digestion, or chemical derivatization. The approach was validated by the comparative analysis of the molecular composition of PCs from human red blood cells. In the total lipid extract of Madin-Darby canine kidney II cells, we detected 46 PC species with unique fatty acid composition and demonstrated that the presence of positional isomers almost doubled the total number of individual molecular species.     

Isolation and identification of 1(3),2-diacylglyceryl-(3)-O-4'-(N,N,N-trimethyl)homoserine from the soil amoeba, Acanthamoeba castellanii

A polar lipid accounting for 12.5% of the total lipid nitrogen has been isolated from the protozoan Acanthamoeba castellanii. On the basis of thin-layer chromatography and mass spectral analysis, the lipid has been identified as diacylglyceryltrimethylhomoserine (DGTS). Fast atom bombardment (FAB) mass spectra of DGTS are reported for the first time and are compared to the FAB mass spectra of phosphatidylcholines and the electron ionization (EI) and field desorption (FD) mass spectra of DGTS. Gas-liquid chromatographic-mass spectrometric (GLC-MS) analysis of the acyl chain composition of this lipid has shown that 87.5% consists of cis-9-octadecenoic acid. Plasma membrane isolated from this organism has shown that labeled DGTS appears in the plasma membrane but is not enriched in this fraction. DGTS has been isolated previously only from a limited number of green plants and one species of fungus. Identification of this lipid in Acanthamoeba indicates that this lipid is distributed among a diverse group of lower eucaryotes.     

Analysis of phospholipid molecular species by liquid chromatography--atmospheric pressure chemical ionisation mass spectrometry of diacylglycerol nicotinates.

A method using liquid chromatography - atmospheric pressure chemical ionisation mass spectrometry was evaluated for determining the molecular species composition of phospholipids (phosphatidylcholines from soybean, egg yolk and bovine liver) after conversion to diacylglycerol nicotinate derivatives. The structures could be deduced from pseudo-molecular ions ([MH-123](+)) and three pairs of monoacyl containing fragment ions. All molecular species in mixed peaks were readily identified and many minor components, earlier not encountered in the samples under investigation, were identified. Acyl chain regioisomers were readily distinguished by the ratio of the [MH-RCHCO](+) ions. Molecular species differing only in the position of the double bonds in one polyunsaturated acyl chain were separated on the basis of retention times. A half quantitative estimation of the molecular species composition of complex samples was achieved by a combination of UV detection and, for mixed peaks, the areas of [MH-123](+) ions.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing iso-branched fatty acids. 1. Differential scanning calorimetric studies

The thermotropic phase behavior of aqueous dispersions of phosphatidylcholines containing one of a series of methyl iso-branched fatty acyl chains was studied by differential scanning calorimetry. These compounds exhibit a complex phase behavior on heating which includes two endothermic events, a gel/gel transition, involving a molecular packing rearrangement between two gel-state forms, and a gel/liquid-crystalline phase transition, involving the melting of the hydrocarbon chains. The gel to liquid-crystalline transition is a relatively fast, highly cooperative process which exhibits a lower transition temperature and enthalpy than do the chain-melting transitions of saturated straight-chain phosphatidylcholines of similar acyl chain length. In addition, the gel to liquid-crystalline phase transition temperature is relatively insensitive to the composition of the aqueous phase. In contrast, the gel/gel transition is a slow process of lower cooperativity than the gel/liquid-crystalline phase transition and is sensitive to the composition of the bulk aqueous phase. The gel/gel transitions of the methyl iso-branched phosphatidylcholines have very different thermodynamic properties and depend in a different way on hydrocarbon chain length than do either the "subtransitions" or the "pretransitions" observed with linear saturated phosphatidylcholines. The gel/gel and gel/liquid-crystalline transitions are apparently concomitant for the shorter chain iso-branched phosphatidylcholines but diverge on the temperature scale with increasing chain length, with a pronounced odd/even alternation of the characteristic temperatures of the gel/gel transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mass spectrometric analysis of long-chain esters of diols

Homologous series of synthetic long-chain monoesters and diesters of 1,2-ethanediol were analyzed by mass spectrometry, and the patterns of fragmentation were studied. Under electron impact saturated ethanediol monoesters yielded prominent ions characteristic of the short-chain diol, such as the rearranged ion formed by 2,3-cleavage (m/e 104) and the ion caused by 3,4-cleavage (m/e 117). Fragments characteristic of the constituent long-chain moieties were the acylium ions [RCO](+), [RCO - 1](+), and the ions [RC(OH)(2)](+). The mass spectra of ethanediol diesters exhibited very intense peaks due to the ions formed by loss of the acyloxy group, [M - RCOO](+), or one carboxylic acid, [M - RCOOH](+). These ions are distinctive for diol diesters. Precise mass measurements by high resolution mass spectrometry verified the composition of the ion fragments. Spectral studies of some monoesters and diesters of 1,3-propanediol, 1,4-butanediol, 2,3-butanediol, and also of some monounsaturated homologues, demonstrated that mass spectrometry is very suitable for the identification, distinction, and analysis of diol lipids.     

Mass-analysed ion kinetic energy spectra and B1E-B2 triple sector mass spectrometric analysis of phosphoinositides by fast atom bombardment

Fast atom bombardment is shown to produce useful spectra of the three phosphoinositides and the metabolically related phospholipids, lysophosphatidylinositol and phosphatidic acid. Analysis of the [M-H]- ions for fatty ester composition by mass-analysed ion kinetic energy spectra (MIKES) is shown to be inadequate to resolve fatty acyl daughter ions when the parent ion contains isobaric species. However, analysis on a triple sector instrument with and without collisional activation does provide complete compositional information. Quantitative analysis of the fatty ester content of each lipid molecular species is complicated by dissimilar ion yields from fatty acyl-bearing fragments from compositionally different parent ions.     

X-ray analysis and calorimetry on phosphatidylcholine model membranes. The influence of length and position of acyl chains upon structure and phase behaviour

The effect of variation of the acyl chain composition of phosphatidylcholines upon thermal behaviour of multilamellar liposomes was evaluated by calorimetry and X-ray studies. A total of thirteen different phosphatidylcholines were examined. They differed from each other in the length as well as in the position of the acyl chains in the glycerol backbone. The experimental results show that the hitherto accepted phase scheme for phosphatidylcholine-water systems is incomplete and has to be extended to include the behaviour of samples that have been stored for long times at low temperatures. The X-ray results show that the structure of the new low-temperature phase is not in agreement with the hexagonal packing of the acyl chains. To explain the X-ray results, a two-dimensional orthorhombic unit cell has to be assumed in order to fit all the observed reflexes in the wide-angle region.     

Computerized Interpretation of the Electron Ionization Mass Spectra of Nucleoside TMS Derivatives

Abstract

A computer program has been developed for the automated interpretation of mass spectra of TMS derivatives of nucleosides found in human urine. The m/z values in the unknown spectrum are compared to m/z values of 3 different ion series commonly observed in the mass spectra of nucleoside TMS derivatives.¹ If a correlation exists, the unknown spectra are marked with color according to the scheme: 1) blue—molecular ion series, 2) red—base ion series and 3) yellow—sugar ion series. The program suggests a structural assignment for each of the marked ions and calculates a series related ion current. The calculated ion current is used to assign the of sugar contained in the unknown nucleoside.     

Ammonia chemical ionization mass spectrometry of lecithins on a gold support

Lecithins directly introduced on a gold wire near the electron beam under ammonia chemical ionization conditions, give mass spectra showing the (M + 1)⁺ ion and ions of structurally significant fragments. Of particular interest is the identification of a substitution-like reaction of ammonia on the head group of the phosphatidylcholines. No instrumental modifications is required.     

Mass spectrometry of sterols. Electron ionization induced fragmentation of C-4-alkylated cholesterols

The electron impact ionization of C-4-alkylated cholest-5-en-3β-hydroxysterols has been investigated. The mass spectra of the C-4-alkylated cholesterols contain a number of ions in the high mass region for which analogous ions are not found in the spectrum of cholesterol. Detailed studies of the composition and origin of these ions have been made by high resolution mass spectrometry and analysis of metastable ions. In addition, a large number of isotopically (deuterium and ¹⁸O) substituted C-4-alkylated analogues have been prepared to assist in the interpretation of the spectra. The combined results indicate the occurrence of a number of very complex and unusual electron ionization induced fragmentations. Most notable of the findings reported herein concerns the demonstration of the formation of an ion involving loss of the elements of ring A with an intramolecular shift of the oxygen and hydrogen atoms of the hydroxyl function to the charge-retaining species.     

Readily synthesized calibration compounds for quadrupole and magnetic instruments for use over the mass range to 2000 daltons

Volatile mass calibration standards have been prepared by esterifying beta-cellobiose (4-beta-D-glucopyranosyl-D-glucose) with a mixture of heptafluorobutyric (HFB) anhydride and pentafluoropropionic (PFP) anhydride. The mixed esters produce spectra that are useful for mass spectrometer calibration with positive or negative ion methane chemical ionization and electron impact over the mass range 300-2000. The spectra contain prominent ions spaced at m/z 20 and m/z 50 intervals. Using the mixed ester with direct insertion probe introduction gives intense spectra that persist for tens of minutes. All signals above m/z 194 derived from these substances disappear rapidly upon withdrawal of the probe. The composition and exact masses are given for the positive and negative ion spectra of a mixed HFB/PFP ester of beta-cellobiose. Two other calibrants are described: one made from beta-cellobiose using a mixture of HFB, PFP and trifluoroacetic anhydrides, and another the HFB/PFP mixed ester of perseitol. These are examples of the flexibility of this approach with respect to mass range and ion composition.     

Isotopologue distributions of peptide product ions by tandem mass spectrometry: quantitation of low levels of deuterium incorporation

Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen, and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, and isotopic labeling by chemical reactions and in studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra of clusters of isotopologue ions obtained in profile mode are fit by nonlinear least squares to a series of Gaussian peaks which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios and obviates the need to determine the intensity of all of the ions of an ID is developed. Consequently a precise and accurate determination of the isotopic composition of a product ion may be obtained from only the initial values of the ID, however, the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy, and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.     

Energy-minimized structures and packing states of a homologous series of mixed-chain phosphatidylcholines: a molecular mechanics study on the diglyceride moieties.

Phosphatidylcholines or C(X):C(Y)PC, quantitatively the most abundant lipids in animal cell membranes, are structurally composed of two parts: a headgroup and a diglyceride. The diglyceride moiety consists of the glycerol backbone and two acyl chains. It is the wide diversity of the acyl chains, or the large variations in X and Y in C(X):C(Y)PC, that makes the family of phosphatidylcholines an extremely complex mixture of different molecular species. Since most of the physical properties of phospholipids with the same headgroup depend strongly on the structures of the lipid acyl chains, the energy-minimized structure and steric energy of each diglyceride moiety of a series of 14 molecular species of phosphatidylcholines with molecular weights identical to that of dimyristoylphosphatidylcholine without the headgroup are determined in this communication by molecular mechanics (MM) calculations. Results of two types of trans-bilayer dimer for each of the 14 molecular species of phosphatidylcholines are also presented; specifically, the dimeric structures are constructed initially based on the partially interdigitated and mixed interdigitated packing motifs followed subsequently by the energy-minimized refinement with MM calculations. Finally, tetramers with various structures to model the lateral lipid-lipid interactions in a lipid bilayer are considered. Results of laborious MM calculations show that saturated diacyl C(X):C(Y)PC with delta C/CL values greater than 0.41 prefer topologically to assemble into tetramers of the mixed interdigitated motif, and those with delta C/CL values less than 0.41 prefer to assemble into tetramers with a repertoire of the partially interdigitated motif. Here, delta C/CL, a lipid asymmetry parameter, is defined as the normalized acyl chain length difference between the sn-1 and sn-2 acyl chains for a C(X):C(Y)PC molecule; an increase in delta C/CL value is an indication of increasing asymmetry between the two lipid acyl chains. These computational results are in complete accord with the calorimetric data presented previously from this laboratory (H-n. Lin, Z-q. Wang, and C. Huang. 1991. Biochim. Biophys. Acta. 1067:17-28).     

On the value of knowing a z* ion for what it is

Computer simulation of database searches of electron transfer dissociation (ETD) spectra using both "bottom up" and "top down" approaches was performed to evaluate the utility of knowing a priori which product ions contain the C-terminus (i.e., the z* ions). In this work, knowledge of the identities of the z* ions was used to exclude putative identifications that are based solely on the mass matching of undifferentiated product ions derived from an experiment with those derived from in silico fragmentation. The benefit from knowing which ions are z* ions was found to be heavily dependent on the quality of the ETD spectra, in terms of sequence coverage afforded by the product ions, the amount of noise in the spectra (i.e., extraneous peaks that do not directly reflect primary structure), and mass measurement accuracy. Under conditions in which the likelihood for misidentifications are high without a priori knowledge of ion types (e.g., b-, y-, c-, or z-ions), a knowledge of which product ions are z* ions allows discrimination against false-positive identifications. Relatively little benefit from knowing which ions are z* ions was noted when product spectra reflected relatively high sequence coverage and when a low fraction of the products ions were due to extraneous peaks (i.e., spectra with relatively little noise). In all cases, specificity is higher with higher mass measurement accuracy with the consequent reduction in benefit from knowledge of which ions are z* ions.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Reaction mechanism and fragment ion structure determination of deprotonated small oligosaccharides, studied by negative ion fast atom bombardment (tandem) mass spectrometry

The negative ion mass spectrometric characteristics of a series of di- and trisaccharides and the tetrasaccharide stachyose have been studied using fast atom bombardment mass spectrometry. The molecular weight of the compounds can easily be derived from their mass spectra, which all show an abundant [M - H]- ion peak. The application of metastable ion and collisional activation techniques to selected pseudomolecular and fragment ions appears to be appropriate for the determination of the position and anomeric type of linkage in the molecules, and provides information concerning the monosaccharide units involved. Important fragmentation reactions have been traced and reaction mechanisms, supported by deuterium labelling experiments, are proposed. An experiment describing the application of the findings of this study to a glycosphingolipid molecule demonstrates its potential value for biological systems.     

Gas chromatographic/mass spectrometric analysis of specific isomers of polychlorodibenzofurans

The gas chromatographic/mass spectrometric characteristics of 26 congeners of polychlorinated dibenzofurans, previously characterized by specific synthetic routes and by standard spectroscopic techniques, have been evaluated. The electron impact mass spectra are not particularly isomer-specific, though 2,3,7,8-tetrachlorodibenzofuran is distinguishable on this basis from the three other tetrachloro isomers investigated in this work. Positive ion methane chemical ionization mass spectra do show a greater degree of isomer distinction, and are reasonably reproducible. Electron attachment negative ion spectral characteristics are also presented. Preliminary results on negative ion chemical ionization mass spectra, obtained using methane plus small amounts of oxygen as reagent gas, are reported.     

Quantitative determination of hydrocarbon chain conformational order in bilayers of saturated phosphatidylcholines of various chain lengths by Fourier transform infrared spectroscopy

The infrared spectra of aqueous dispersions of a homologous series of symmetric-chain, disaturated phosphatidylcholines, with fatty acyl chain lengths ranging from 12 to 19 carbons, have been measured at comparable reduced temperatures in their liquid-crystalline phases. The infrared spectra of these compounds contain bands that are dependent on the conformation of the fatty acyl chains. In particular, in the 1400-1300-cm-1 spectral region, there are bands due to CH2 wagging which are specific for the different types of gauche conformers. Thus, gauche-trans-gauché sequences (or kinks) give a band at 1367 cm-1, end-gauche conformers a band at 1341 cm-1, and double-gauche conformers a band at 1355 cm-1. The intensities of these bands were determined and normalized to the intensity of the conformation-insensitive band due to symmetric methyl bending at 1378 cm-1. The intensities of the different "gauche" bands yield a "per chain" intensity, which is directly related to the concentration of the different types of conformational defects. We find that, within experimental error, the concentration of end-gauche and double-gauche conformers is relatively low and practically invariant with chain length when a series of homologous phosphatidylcholines are compared at the same reduced temperature. In contrast, the concentration of gauche-trans-gauché sequences (kink defects) is much higher and increases as the chain length increases. For dipalmitoylphosphatidylcholine we find that there are about 1.2 kink, 0.5-0.6 end-gauche, and 0.4 double-gauche conformers per hydrocarbon chain.(ABSTRACT TRUNCATED AT 250 WORDS)