Search

Cardiolipin (CL) analysis demands high specificity, due to the extensive diversity of CL structures, and high sensitivity, due to their low relative abundance within the lipidome. While electrospray ionization mass spectrometry (ESI-MS) is the most widely used technology in lipidomics, the potential for multiple charging presents unique challenges for CL identification and quantification. Depending on the conditions, ESI-MS of lipid extracts in negative ion mode can give rise to cardiolipins ionized as both singly and doubly deprotonated anions. This signal degeneracy diminishes the signal-to-noise ratio, while in addition (for direct infusion), the dianion population falls within a m / z range already heavily congested with monoanions from more abundant glycerophospholipid subclasses. Herein, we describe a direct infusion strategy for CL profiling from total lipid extracts utilizing gas-phase proton-transfer ion/ion reactions. In this approach, lipid extracts are ionized by negative ion ESI generating both singly deprotonated phospholipids and doubly deprotonated CL anions. Charge reduction of the negative ion population by ion/ion reactions leads to an enhancement in singly deprotonated [CL - H] - species via proton transfer to the corresponding [CL - 2H] 2- ̅ dianions. To concentrate the [CL - H] - anion signal, multiple iterations of ion accumulation and proton-transfer ion/ion reaction can be performed prior to subsequent interrogation. Mass selection and collisional activation of the enriched population of [CL - H] - anions facilitates the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Demonstrated advantages of this new approach derive from the improved performance in complex mixture analysis affording detailed characterization of low abundant CLs directly from a total biological extract.

Representing the most fundamental lipid class, fatty acids (FA) play vital biological roles serving as energy sources, cellular signaling molecules, and key architectural components of complex lipids. Direct infusion electrospray ionization spectrometry, also known as shotgun lipidomics, has emerged as a rapid and powerful toolbox for lipid analysis. While shotgun lipidomics can be a sensitive approach to FA detection, the diverse molecular structure of FA presents challenges for unambiguous identification and the relative quantification of isomeric contributors. In particular, pinpointing double bond position(s) in unsaturated FA and determining the relative contribution of double bond isomers has limited the application of the shotgun approach. Recently, we reported the use of gas-phase ion/ion reactions to facilitate the identification of FA. Briefly, singly deprotonated FA anions undergo charge inversion when reacted in the gas phase with tris-phenanthroline magnesium dications by forming [FA - H + MgPhen] + complex ions. These charge-inverted FA complex cations fragment upon ion-trap collision-induced dissociation (CID) to generate product ion spectra unique to individual FA isomers. Herein, we report the development of a mass spectral library comprised of [FA - H + MgPhen] + product ion spectra. The developed FA library permits confident FA identification, including polyunsaturated FA isomers. Furthermore, we demonstrate the ability to determine relative contributions of isomeric FA using multiple linear regression analysis paired with gas-phase ion/ion reactions. We successfully applied the presented method to generate a FA profile for bovine liver phospholipidome based entirely on gas-phase chemistries.

Fatty acids (FA) play vital biological roles as energy sources, signaling molecules and key building blocks of complex lipids in cell membranes. Modifications to FA structure and composition are associated with the onset and progression of a number of chronic diseases. Consequently, the sensitive detection and unambiguous structure elucidation of FA is integral to the advancement of biomedical sciences. Recent advances in FA analysis have taken advantage of wet chemical derivatization to enhance detection and drive unique fragmentation in tandem mass spectrometry protocols. Here, we significantly further this approach through demonstrating gas-phase charge inversion of singly deprotonated FA ions, [M - H] - , using doubly charged tris-phenanthroline alkaline earth metal complexes, [Cat(Phen) 3 ] 2+ (Cat = Mg 2+ , Ca 2+ , Sr 2+ , or Ba 2+ ). Metal cationized FA, [M - H + Cat] + are obtained after the gas-phase ion/ion reaction. Low-energy collision-induced dissociation (CID) of the [M - H + Cat] + cations facilitates double bond localization for a variety of monounsaturated and polyunsaturated FAs. Ultimately, detailed characterization presented unambiguous distinction among FA double bond positional isomers, such as n-3 and n-6 isomers. The method was successfully used to identify the FA profile of corn oil, including double bond localization for unsaturated FAs present.

Complex mixtures of plant derived triglycerol (TG) lipids are commonly used as feedstock components for the production of industrial polymers. However, there remains a need for the development of analytical strategies to investigate the intrinsic intermolecular cross-linking reactivity of individual TG molecules within these mixtures as a function of their structures and physicochemical properties, and for the characterization of the resultant products. Here, to address this need, we describe a novel multistage tandem mass spectrometry based method for intermolecular cross-linking and subsequent structural characterization of TG lipid ions in the gas phase. Cross-linking reactions were initiated using 266 nm ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) of saturated or unsaturated TG dimers introduced via electrospray ionization into a linear ion trap mass spectrometer as noncovalent complexes with protonated 3,4-, 2,4- or 3,5- diiodoaniline (diIA). UVPD resulted in the initial formation of an anilinyl biradical via the sequential loss of two iodine radicals, which underwent further reaction to yield multiple cross-linked TG products along with competing noncross-linking processes. These chemistries are proposed to occur via sequential combinations of hydrogen abstraction (H-abstraction), radical addition and radical recombination. Multistage collision induced dissociation tandem mass spectrometry (CID-MS(n)) was used to obtain evidence for the structures and mechanisms of formation for these products, as a function of both the TG lipid and diIA ion structures. The efficiency of the UVPD reaction was shown to be dependent on the number of unsaturation sites present within the TG lipids. However, when unsaturation sites were present, formation of the cross-linked and noncross-linked product ions via H-abstraction and radical addition mechanisms was found to be competitive. Finally, the identity of the anilinyl biradical (e.g., 3,4- versus 2,4-substituted) was found to significantly affect the distribution of these two types of product ions. Importantly, owing to the observed propensity for cross-linking to occur via H-abstraction-initiated processes, this novel gas-phase cross-linking reaction provides a convenient method to link two molecules covalently without the requirement of any specific functional group, and therefore could be applied to examine the gas-phase intermolecular interactions and cross-linking of a wide range of biomolecular classes.

Aromatic radicals form in a variety of reacting gas-phase systems, where their molecular weight growth reactions with unsaturated hydrocarbons are of considerable importance. We have investigated the ion-molecule reaction of the aromatic distonic N-methyl-pyridinium-4-yl (NMP) radical cation with 2-butyne (CH(3)C≡CCH(3)) using ion trap mass spectrometry. Comparison is made to high-level ab initio energy surfaces for the reaction of NMP and for the neutral phenyl radical system. The NMP radical cation reacts rapidly with 2-butyne at ambient temperature, due to the apparent absence of any barrier. The activated vinyl radical adduct predominantly dissociates via loss of a H atom, with lesser amounts of CH(3) loss. High-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry allows us to identify small quantities of the collisionally deactivated reaction adduct. Statistical reaction rate theory calculations (master equation/RRKM theory) on the NMP+2-butyne system support our experimental findings, and indicate a mechanism that predominantly involves an allylic resonance-stabilized radical formed via H atom shuttling between the aromatic ring and the C(4) side-chain, followed by cyclization and/or low-energy H atom β-scission reactions. A similar mechanism is demonstrated for the neutral phenyl radical (Ph˙)+2-butyne reaction, forming products that include 3-methylindene. The collisionally deactivated reaction adduct is predicted to be quenched in the form of a resonance-stabilized methylphenylallyl radical. Experiments using a 2,5-dichloro substituted methyl-pyridiniumyl radical cation revealed that in this case CH(3) loss from the 2-butyne adduct is favoured over H atom loss, verifying the key role of ortho H atoms, and the shuttling mechanism, in the reactions of aromatic radicals with alkynes. As well as being useful phenyl radical analogues, pyridiniumyl radical cations may form in the ionosphere of Titan, where they could undergo rapid molecular weight growth reactions to yield polycyclic aromatic nitrogen hydrocarbons (PANHs).

α-Carboxylate radical anions are potential reactive intermediates in the free radical oxidation of biological molecules (e.g., fatty acids, peptides and proteins). We have synthesised well-defined α-carboxylate radical anions in the gas phase by UV laser photolysis of halogenated precursors in an ion-trap mass spectrometer. Reactions of isolated acetate (˙CH(2)CO(2)(-)) and 1-carboxylatobutyl (CH(3)CH(2)CH(2)˙CHCO(2)(-)) radical anions with dioxygen yield carbonate (CO(3)˙(-)) radical anions and this chemistry is shown to be a hallmark of oxidation in simple and alkyl-substituted cross-conjugated species. Previous solution phase studies have shown that C(α)-radicals in peptides, formed from free radical damage, combine with dioxygen to form peroxyl radicals that subsequently decompose into imine and keto acid products. Here, we demonstrate that a novel alternative pathway exists for two α-carboxylate C(α)-radical anions: the acetylglycinate radical anion (CH(3)C(O)NH˙CHCO(2)(-)) and the model peptide radical anion, YGGFG˙(-). Reaction of these radical anions with dioxygen results in concerted loss of carbon dioxide and hydroxyl radical. The reaction of the acetylglycinate radical anion with dioxygen reveals a two-stage process involving a slow, followed by a fast kinetic regime. Computational modelling suggests the reversible formation of the C(α) peroxyl radical facilitates proton transfer from the amide to the carboxylate group, a process reminiscent of, but distinctive from, classical proton-transfer catalysis. Interestingly, inclusion of this isomerization step in the RRKM/ME modelling of a G3SX level potential energy surface enables recapitulation of the experimentally observed two-stage kinetics.

The gas phase degradation reactions of the chemical warfare agent (CWA) simulant, dimethyl methylphosphonate (DMMP), with the hydroperoxide anion (HOO(-)) were investigated using a modified quadrupole ion trap mass spectrometer. The HOO(-) anion reacts readily with neutral DMMP forming two significant product ions at m/z 109 and m/z 123. The major reaction pathways correspond to (i) the nucleophilic substitution at carbon to form [CH(3)P(O)(OCH(3))O](-) (m/z 109) in a highly exothermic process and (ii) exothermic proton transfer. The branching ratios of the two reaction pathways, 89% and 11% respectively, indicate that the former reaction is significantly faster than the latter. This is in contrast to the trend for the methoxide anion with DMMP, where proton transfer dominates. The difference in the observed reactivities of the HOO(-) and CH(3)O(-) anions can be considered as evidence for an alpha-effect in the gas phase and is supported by electronic structure calculations at the B3LYP/aug-cc-pVTZ//B3LYP/6-31+G(d) level of theory that indicate the S(N)2(carbon) process has an activation energy 7.8 kJ mol(-1) lower for HOO(-) as compared to CH(3)O(-). A similar alpha-effect was calculated for nucleophilic addition-elimination at phosphorus, but this process--an important step in the perhydrolysis degradation of CWAs in solution--was not observed to occur with DMMP in the gas phase. A theoretical investigation revealed that all processes are energetically accessible with negative activation energies. However, comparison of the relative Arrhenius pre-exponential factors indicate that substitution at phosphorus is not kinetically competitive with respect to the S(N)2(carbon) and deprotonation processes.

Proton-bound dimers consisting of two glycerophospholipids with different headgroups were prepared using negative ion electrospray ionization and dissociated in a triple quadrupole mass spectrometer. Analysis of the tandem mass spectra of the dimers using the kinetic method provides, for the first time, an order of acidity for the phospholipid classes in the gas phase of PE < PA << PG < PS < PI. Hybrid density functional calculations on model phospholipids were used to predict the absolute deprotonation enthalpies of the phospholipid classes from isodesmic proton transfer reactions with phosphoric acid. The computational data largely support the experimental acidity trend, with the exception of the relative acidity ranking of the two most acidic phospholipid species. Possible causes of the discrepancy between experiment and theory are discussed and the experimental trend is recommended. The sequence of gas phase acidities for the phospholipid headgroups is found to (1) have little correlation with the relative ionization efficiencies of the phospholipid classes observed in the negative ion electrospray process, and (2) correlate well with fragmentation trends observed upon collisional activation of phospholipid [M - H](-) anions.