Ozonolysis of phospholipid double bonds during electrospray ionization : A new tool for structure determination

Phospholipids are the key structural component of cell membranes, and recent advances in electrospray ionization mass spectrometry provide for the fast and efficient analysis of these compounds in biological extracts.1-3 The application of electrospray ionization tandem mass spectrometry (ESI-MS/MS) to phospholipid analysis has demonstrated several key advantages over the more traditional chromatographic methods, including speed and greater structural information.4 For example, the ESI-MS/MS spectrum of a typical phospholipidsparticularly in negative ion modesreadily identifies the carbon chain length and the degree of unsaturation of each of the fatty acids esterified to the parent molecule.5 A critical limitation of conventional ESI-MS/MS analysis, however, is the inability to uniquely identify the position of double bonds within the fatty acid chains. This is especially problematic given the importance of double bond position in determining the biological function of lipid classes.6 Previous attempts to identify double bond position in intact phospholipids using mass spectrometry employ either MS3 or offline chemical derivatization.7-11 The former method requires specialized instrumentation and is rarely applied, while the latter methods suffer from complications inherent in sample handling prior to analysis. In this communication we outline a novel on-line approach for the identification of double bond position in intact phospholipids. In our method, the double bond(s) present in unsaturated phospholipids are cleaved by ozonolysis within the ion source of a conventional ESI mass spectrometer to give two chemically induced fragment ions that may be used to unambiguously assign the position of the double bond. This is achieved by using oxygen as the electrospray nebulizing gas in combination with high electrospray voltages to initiate the formation of an ozoneproducing.

A combined neutralization-reionization mass spectrometric and theoretical study of oxyallyl and other elusive [C3,H4,O] neutrals

Five different anionic [C3′H4′O]•- isomers, i.e. the radical anions of acrolein, acetyl carbene, formyl methyl carbene, methoxy vinylidene, and oxyallyl are generated in an ion beam mass spectrometer and subjected to neutralization-reionization (NR) mass spectrometric experiments including neutral and ion decomposition difference (NIDD) mass spectrometry; the latter allows for the examination of the neutrals' unimolecular reactivity. Further, the anionic, the singlet and triplet neutral, and the cationic [C3′H4′O] •-/0/•+ potentialenergy surfaces are calculated at the B3LYP/6-311++G(d,p) level of theory. For some species, notably the singlet state of oxyallyl, the theoretical treatment is complemented by G2, CASSCF, and MR-CI calculations. Theory and experiment are in good agreement in that at the neutral stage (i) acrolein does not react within the μsec timescale, (ii) acetyl and formyl methyl carbenes isomerize to methyl ketene, (iii) methoxy vinylidene rearranges to methoxy acetylene, (iv) singlet 1A1 oxyallyl undergoes ring closure to cyclopropanone, and (v) triplet 3B2 oxyallyl may have a lifetime sufficient to survive a NR experiment.

Polarized infrared absorption spectrum of matrix-isolated methylperoxyl radicals, CH3OO (X)over-tilde 2A ''

We have used a tandem pair of supersonic nozzles to produce clean samples of CH3OO radicals in cryogenic matrices. One hyperthermal nozzle decomposes azomethane (CH3NNCH3) to generate intense pulses of CH3 radicals, While the second nozzle alternately fires a burst Of O-2/Ar at the 20 K matrix. The CH3/O-2/20 K argon radical sandwich acts to produce target methylperoxyl radicals: CH3 + O-2 --> CH3OO. The absorption spectra of the radicals are monitored with a Fourier transform infrared spectrometer. We report 10 of the 12 fundamental infrared bands of the methylperoxyl radical CH3OO, (X) over tilde (2)A", in an argon matrix at 20 K. The experimental frequencies (cm(-1)) and polarizations follow: the a' modes are 3032, 2957, 1448, 1410, 1180, 1109, 90, 492, while the a" modes are 3024 and 1434. We cannot detect the asymmetric CH3 rocking mode, nu(11), nor the torsion, nu(12). The infrared spectra of (CH3OO)-O-18-O-18, (CH3OO)-C-13, and CD3OO have been measured as well in order to determine the isotopic shifts. The experimental frequencies, {nu}, for the methylperoxyl radicals are compared to harmonic frequencies, {omega}, resulting from a UB3LYP/6-311G(d,p) electronic structure calculation. Linear dichroism spectra were measured with photooriented radical samples in order to establish the experimental polarizations of most vibrational bands. The methylperoxyl radical matrix frequencies listed above are within +/-2% of the gas-phase vibrational frequencies. A final set of vibrational frequencies for the H radical, are recommended. See also http://ellison.colorado.edu/methylperoxyl.

Mechanisms of glycerol dehydration

Dehydration of neutral and protonated glycerol was investigated using quantum mechanical calculations (CBS-QB3). Calculations on neutral glycerol show that there is a high barrier for simple 1,2-dehydration, E-a = 70.9 kcal mol(-1), which is lowered to 65.2 kcal mol(-1) for pericyclic 1,3-dehydration. In contrast, the barriers for dehydration of protonated glycerol are much lower. Dehydration mechanisms involving hydride transfer, pinacol rearrangement, or substitution reactions have barriers between 20 and 25 kcal mol(-1). Loss of water from glycerol via substitution results in either oxirane or oxetane intermediates, which can interconvert over a low barrier. Subsequent decomposition of these intermediates proceeds via either a second dehydration step or loss of formaldehyde. The computed mechanisms for decomposition of protonated glycerol are supported by the gas-phase fragmentation of protonated glycerol observed using a triple-quadrupole mass spectrometer.

Ozone-Induced Dissociation on a Modified Tandem Linear Ion-Trap : Observations of Different Reactivity for Isomeric Lipids

Ozone-induced dissociation (OzID) exploits the gas-phase reaction between mass-selected lipid ions and ozone vapor to determine the position(s) of unsaturation In this contribution, we describe the modification of a tandem linear ion-trap mass spectrometer specifically for OzID analyses wherein ozone vapor is supplied to the collision cell This instrumental configuration provides spatial separation between mass-selection, the ozonolysis reaction, and mass-analysis steps in the OzID process and thus delivers significant enhancements in speed and sensitivity (ca 30-fold) These improvements allow spectra revealing the double-bond position(s) within unsaturated lipids to be acquired within 1 s significantly enhancing the utility of OzID in high-throughput lipidomic protocols The stable ozone concentration afforded by this modified instrument also allows direct comparison of relative reactivity of isomeric lipids and reveals reactivity trends related to (1) double-bond position, (2) substitution position on the glycerol backbone, and (3) stereochemistry For cis- and trans-isomers, differences were also observed in the branching ratio of product ions arising from the gas-phase ozonolysis reaction, suggesting that relative ion abundances could be exploited as markers for double-bond geometry Additional activation energy applied to mass-selected lipid ions during injection into the collision cell (with ozone present) was found to yield spectra containing both OzID and classical-CID fragment ions This combination CID-OzID acquisition on an ostensibly simple monounsaturated phosphatidylcholine within a cow brain lipid extract provided evidence for up to four structurally distinct phospholipids differing in both double-bond position and sn-substitution U Am Soc Mass Spectrom 2010, 21, 1989-1999) (C) 2010 American Society for Mass Spectrometry

The loss of CO from the ortho, meta and para forms of deprotonated methyl benzoate in the gas phase

The ortho, meta and para anions of methyl benzoate may be made in the source of a mass spectrometer by the S(N)2(Si) reactions between HO- and methyl (o-, m-, and p-trimethylsilyl)benzoate respectively. All three anions lose CO upon collisional activation to form the ortho anion of anisole in the ratio ortho>>meta > para. The rearrangement process is charge directed through the ortho anion. Theoretical calculations at the B3LYP/6-311++G(d,p)//HF/6-31+G(d) level of theory indicate that the conversion of the meta and para anions to the ortho anion prior to loss of CO involve 1,2-H transfer(s), rather than carbon scrambling of the methoxycarbonylphenyl anion. There are two mechanisms which can account for this rearrangement, viz. (A) cyclisation of the ortho anion centre to the carbonyl group of the ester to give a cyclic carbonyl system in which the incipient methoxide anion substitutes at one of the two equivalent ring carbons of the three membered ring to yield an intermediate which loses CO to give the ortho anion of anisole, and (B) an elimination reaction to give an intermediate benzyne-methoxycarbonyl anion complex in which the MeOCO- species acts as a MeO- donor, which then adds to benzyne to yield the ortho anion of anisole. Calculations at the B3LYP/6-311++G(d,p)//HF/6-31+G(d) level of theory indicate that (i) the barrier in the first step (the rate determining step) of process A is 87 kJ mol(-1) less than that for the synchronous benzyne process B, and (ii) there are more low frequency vibrations in the transition state for benzyne process B than for the corresponding transition state for process A. Stepwise process A has the lower barrier for the rate determining step, and the lower Arrhenius factor: we cannot differentiate between these two mechanisms on available evidence.

Imaging of human lens lipids by desorption electrospray ionization mass spectrometry

The lipid composition of the human lens is distinct from most other tissues in that it is high in dihydrosphingomyelin and the most abundant glycerophospholipids in the lens are unusual 1-O-alkyl-ether linked phosphatidylethanolamines and phosphatidylserines. In this study, desorption electrospray ionization (DESI) mass spectrometry-imaging was used to determine the distribution of these lipids in the human lens along with other lipids including, ceramides, ceramide-1-phosphates, and lyso 1-O-alkyl ethers. To achieve this, 25 μm lens slices were mounted onto glass slides and analyzed using a linear ion-trap mass spectrometer equipped with a custom-built, 2-D automated DESI source. In contrast to other tissues that have been previously analyzed by DESI, the presence of a strong acid in the spray solvent was required to desorb lipids directly from lens tissue. Distinctive distributions were observed for [M + H]+ ions arising from each lipid class. Of particular interest were ionized 1-O-alkyl phosphatidylethanolamines and phosphatidylserines, PE (18:1e/18:1), and PS (18:1e/18:1), which were found in a thin ring in the outermost region of the lens. This distribution was confirmed by quantitative analysis of lenses that were sectioned into four distinct regions (outer, barrier, inner, and core), extracted and analyzed by electrospray ionization tandem mass spectrometry. DESI-imaging also revealed a complementary distribution for the structurally-related lyso 1-O-alkyl phosphatidylethanolamine, LPE (18:1e), which was localized closer to the centre of the lens. The data obtained in this study indicate that DESI-imaging is a powerful tool for determining the spatial distribution of human lens lipids. © 2010 American Society for Mass Spectrometry.

Conversion of linear to rhombic C-4 in the gas phase: A joint experimental and theoretical study

Charge reversal (CR) and neutralization reionization (NR) experiments carried out on a 4-sector mass spectrometer demonstrate that isotopically labeled, linear C-4 anion rearranges upon collisional oxidation. The cations and neutrals formed in these experiments exhibit differing degrees of isotopic scrambling in their fragmentation patterns, indicative of (at least) partial isomerization of both states. Theoretical studies, employing the CCSD(T)/aug-cc-pVDZ//B3LYP/6-31G(d) level of theory, favor conversion to the rhombic C-4 isomer on both cationic and neutral potential-energy surfaces with the rhombic structures predicted to be slightly more stable than the linear forms in each case. The combination of experiment with theory indicates that the elusive rhombic C-4 is formed as a cation and as a neutral following charge stripping of linear C-4(-)

Syntheses of NCN and NC3N from ionic precursors in the gas phase and an unusual rearrangement of neutral NC3N : A joint experimental and theoretical study

Neutral NCN is made in a mass spectrometer by charge stripping of NCN-., while neutral dicyanocarbene NCCCN can be formed by neutralization of either the corresponding anionic and cationic species, NCCCN-. and NCCCN+.. Theoretical calculations at the RCCSD(T)/aug-cc-pVTZ//B3LYP/6-31+G(d) level of theory indicate that the (3)Sigma (-)(g) State of NCCCN is 18 kcal mol(-1) more stable than the (1)A(1) state. While the majority of neutrals formed from either NCCCN-. or NCCCN+. correspond to NCCCN, a proportion of the neutral NCCCN molecules have sufficient excess energy to effect rearrangement, as evidenced by a loss of atomic carbon in the neutralization reionization (NR) spectra of either NCCCN+. and NCCCN-.. C-13 labeling studies indicate that loss of carbon occurs statistically following or accompanied by scrambling of all three carbon atoms. A theoretical study at the B3LYP/6-31+G(d)//B3LYP/6-31+G(d) level of theory indicates that C loss is a consequence of the rearrangement sequence NCCCN --> CNCCN --> CNCNC and that C scrambling occurs within singlet CNCCN via the intermediacy of a four-membered C-2v-symmetrical transition structure.

Formation of neutral C7H2 isomers from pour isomeric C7H2 radical anion precursors in the gas phase

Consideration of theoretical calculations \[E3LYP/aug-cc-pVDZ//B3LYP/6-31G(d)\] of the structures of ten C7H2 neutral isomers and the nine corresponding C7H2 radical anions have led us to synthesize four stable C7H2 radical anions in the ion source of our ZAB 2HF mass spectrometer, and to convert these to C7H2 neutrals. The four radical anion isomers prepared were (i) \[(HC equivalent to C)(2)C=C=C\](-.) \[from the reaction between (HC equivalent to C)(3)COCH3 and HO- \], (ii) \[HC=C=C=C=C=C=CH\](-.) \[from the reaction between HC equivalent to C-C equivalent to C-CD(OH)-C equivalent to CH and HO-\], (iii) \[C=C=C=C=C=C=CH2\](-.) \[from the reaction between DC equivalent to C-C equivalent to C-C equivalent to C-CH2OCH2CH3 and HO-\], and (iv) \[C equivalent to C-CH2-C equivalent to C-C equivalent to C\](-.) \[from the bis desilylation reaction of (CH3)(3)Si-C equivalent to C-CH2-C equivalent to C-C equivalent to C-Si (CH3)(3)With SF6-.\]. The four anions were further characterized by their collisional activation (negative ion) and charge reversal (CR, positive ion) mass spectra. The anions were converted into their corresponding neutrals by charge stripping, and the correspondence between the charge reversal (CR) and neutralization reionization (-NR+) mass spectra of each anion is taken as evidence that within the time frame of the -NR+ experiment (some 10(-6) s), each neutral is stable and undergoes no major rearrangement or interconversion to a more stable isomer. Theory and experiment are in accord for these systems.

Formation of neutral C7H2 isomers from four isomeric C7H2 radical anion precursors in the gas phase

Consideration of theoretical calculations [B3LYP/aug-cc-pVDZ//B3LYP/6-31G(d)] of the structures of ten C7H2 neutral isomers and the nine corresponding C7H2 radical anions have led us to synthesize four stable C7H2 radical anions in the ion source of our ZAB 2HF mass spectrometer, and to convert these to C7H2 neutrals. The four radical anion isomers prepared were (i) [(HC≡C)2C=C=C]-̇ [from the reaction between (HC≡C)3COCH3 and HO- ], (ii) [HC=C=C=C=C=C=CH]-̇ [from the reaction between HC≡C-C≡C- CD(OH)-C≡CH and HO-], (iii) [C=C=C=C=C=C=CH2]-̇ [from the reaction between DC≡C-C≡C- C≡C-CH2OCH2CH3 and HO-], and (iv) [C≡C-CH2-C≡C-C≡C]-̇ [from the bis desilylation reaction of (CH3)3Si-C≡C-CH2-C≡C-C≡C-Si (CH3)3 with SF6 -̇]. The four anions were further characterized by their collisional activation (negative ion) and charge reversal (CR, positive ion) mass spectra. The anions were converted into their corresponding neutrals by charge stripping, and the correspondence between the charge reversal (CR) and neutralization reionization (-NR+) mass spectra of each anion is taken as evidence that within the time frame of the -NR+ experiment (some 10-6 s), each neutral is stable and undergoes no major rearrangement or interconversion to a more stable isomer. Theory and experiment are in accord for these systems.

Investigation of the gas phase reactivity of the 1-adamantyl radical using a distonic radical anion approach

The gas phase reactions of the bridgehead 3-carboxylato-1-adamantyl radical anion were observed with a series of neutral reagents using a modified electrospray ionisation linear ion trap mass spectrometer. This distonic radical anion was observed to undergo processes suggestive of radical reactivity including radical-radical combination reactions, substitution reactions and addition to carbon-carbon double bonds. The rate constants for reactions of the 3-carboxylato-1-adamantyl radical anion with the following reagents were measured ( in units 10(-12) cm(3) molecule(-1) s(-1)): O-18(2) ( 85 +/- 4), NO ( 38.4 +/- 0.4), I-2 ( 50 +/- 50), Br-2 ( 8 +/- 2), CH3SSCH3 ( 12 +/- 2), styrene ( 1.20 +/- 0.03), CHCl3 ( H abstraction 0.41 +/- 0.06, Cl abstraction 0.65 +/- 0.1), CDCl3 ( D abstraction 0.035 +/- 0.01, Cl abstraction 0.723 +/- 0.005), allyl bromide (Br abstraction 0.53 +/- 0.04, allylation 0.25 +/- 0.01). Collision rates were calculated and reaction efficiencies are also reported. This study represents the first quantitative measurement of the gas phase reactivity of a bridgehead radical and suggests that distonic radical anions are good models for the study of their elusive uncharged analogues.

Fragmentations of deprotonated alkyl hydroperoxides (ROO-) upon collisional activation : A combined experimental and computational study

The collision-induced dissociation ( CID) mass spectra of the \[M-H](-) anions of methyl, ethyl, and tert-butyl hydroperoxides have been measured over a range of collision energies in a flowing afterglow - selected ion flow tube (FA-SIFT) mass spectrometer. Activation of the CH3OO- anion is found to give predominantly HO- fragment anions whilst CH3CH2OO- and (CH3)(3)COO- produce HOO- as the major ionic fragment. These results, and other minor fragmentation pathways, can be rationalized in terms of unimolecular rearrangement of the activated anions with subsequent decomposition. The rearrangement reactions occur via initial abstraction of a proton from the alpha-carbon in the case of CH3OO- or the beta-carbon for CH3CH2OO- and (CH3)(3)COO-. Electronic structure calculations suggest that for the CH3CH2OO- anion, which can theoretically undergo both alpha- and beta-proton abstraction, the latter pathway, resulting in HOO- + CH2CH2, is energetically preferred.

Generation of three isomers of C7H- in the gas phase. A joint experimental and theoretical study

Three anion isomers of formula C7H have been synthesised in the mass spectrometer by unequivocal routes. The structures of the isomers are \[HCCC(C-2)(2)](-), C6CH- and C2CHC4-. One of these, \[HCCC(C-2)(2)](-), is formed in sufficient yield to allow it to be charge stripped to the corresponding neutral radical.

Structural identification of hindered amine light stabilisers in coil coatings using electrospray ionisation tandem mass spectrometry

Hindered amine light stabilisers (HALS) are the most effective antioxidants currently available for polymer systems in post-production, in-service applications, yet the mechanism of their action is still not fully understood. Structural characterisation of HALS in polymer matrices, particularly the identification of structural modifications brought about by oxidative conditions, is critical to aid mechanistic understanding of the prophylactic effects of these molecules. In this work, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was applied to the analysis of a suite of commercially available 2,2,6,6-tetramethylpiperidine-based HALS. Fragmentation mechanisms for the \[M + H](+) ions are proposed, which provide a rationale for the product ions observed in the MS/MS and MS(3) mass spectra of N-H, N-CH(3), N-C(O)CH(3) and N-OR containing HALS (where R is an alkyl substituent). A common product ion at m/z 123 was identified for the group of antioxidants containing N-H, N-CH3 or N-C(0)CH3 functionality, and this product ion was employed in precursor ion scans on a triple quadrupole mass spectrometer to identify the HALS species present in a crude extract from of a polyester-based coil coating. Using MS/MS, two degradation products were unambiguously identified. This technique provides a simple and selective approach to monitoring HALS structures within complex matrices. Copyright (C) 2010 John Wiley & Sons, Ltd.