Alan T. Maccarone

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

Ozone-induced dissociation of conjugated lipids reveals significant reaction rate enhancements and characteristic odd-electron product ions

AbstractOzone-induced dissociation (OzID) is an alternative ion activation method that relies on the gas phase ion-molecule reaction between a mass-selected target ion and ozone in an ion trap mass spectrometer. Herein, we evaluated the performance of OzID for both the structural elucidation and selective detection of conjugated carbon-carbon double bond motifs within lipids. The relative reactivity trends for [M + X]+ ions (where X = Li, Na, K) formed via electrospray ionization (ESI) of conjugated versus nonconjugated fatty acid methyl esters (FAMEs) were examined using two different OzID-enabled linear ion-trap mass spectrometers. Compared with nonconjugated analogues, FAMEs derived from conjugated linoleic acids were found to react up to 200 times faster and to yield characteristic radical cations. The significantly enhanced reactivity of conjugated isomers means that OzID product ions can be observed without invoking a reaction delay in the experimental sequence (i.e., trapping of ions in the presence of ozone is not required). This possibility has been exploited to undertake neutral-loss scans on a triple quadrupole mass spectrometer targeting characteristic OzID transitions. Such analyses reveal the presence of conjugated double bonds in lipids extracted from selected foodstuffs. Finally, by benchmarking of the absolute ozone concentration inside the ion trap, second order rate constants for the gas phase reactions between unsaturated organic ions and ozone were obtained. These results demonstrate a significant influence of the adducting metal on reaction rate constants in the fashion Li > Na > K.

Direct Observation of Photodissociation Products from Phenylperoxyl Radicals Isolated in the Gas Phase

Gas phase peroxyl radicals are central to our chemical understanding of combustion and atmospheric processes and are typically characterized by strong absorption in the UV (λ(max) ≈ 240 nm). The analogous maximum absorption feature for arylperoxyl radicals is predicted to shift to the visible but has not previously been characterized nor have any photoproducts arising from this transition been identified. Here we describe the controlled synthesis and isolation in vacuo of an array of charge-substituted phenylperoxyl radicals at room temperature, including the 4-(N,N,N-trimethylammonium)methyl phenylperoxyl radical cation (4-Me3N([+])CH2-C6H4OO(•)), using linear ion-trap mass spectrometry. Photodissociation mass spectra obtained at wavelengths ranging from 310 to 500 nm reveal two major photoproduct channels corresponding to homolysis of aryl-OO and arylO-O bonds resulting in loss of O2 and O, respectively. Combining the photodissociation yields across this spectral window produces a broad (FWHM ≈ 60 nm) but clearly resolved feature centered at λ(max) = 403 nm (3.08 eV). The influence of the charge-tag identity and its proximity to the radical site are investigated and demonstrate no effect on the identity of the two dominant photoproduct channels. Electronic structure calculations have located the vertical B ← X transition of these substituted phenylperoxyl radicals within the experimental uncertainty and further predict the analogous transition for unsubstituted phenylperoxyl radical (C6H5OO(•)) to be 457 nm (2.71 eV), nearly 45 nm shorter than previous estimates and in good agreement with recent computational values.

Elucidating the chemical structure of native 1-deoxysphingosine

The 1-deoxysphingolipids (1-deoxySLs) are formed by an alternate substrate usage of the enzyme, serine-palmitoyltransferase, and are devoid of the C1-OH-group present in canonical sphingolipids. Pathologically elevated 1-deoxySL levels are associated with the rare inherited neuropathy, HSAN1, and diabetes type 2 and might contribute to β cell failure and the diabetic sensory neuropathy. In analogy to canonical sphingolipids, it was assumed that 1-deoxySLs also bear a (4E) double bond, which is normally introduced by sphingolipid delta(4)-desaturase 1. This, however, was never confirmed. We therefore supplemented HEK293 cells with isotope-labeled D3-1-deoxysphinganine and compared the downstream formed D3-1-deoxysphingosine (1-deoxySO) to a commercial synthetic SPH m18:1(4E)(3OH) standard. Both compounds showed the same m/z, but differed in their RPLC retention time and atmospheric pressure chemical ionization in-source fragmentation, suggesting that the two compounds are structural isomers. Using dimethyl disulfide derivatization followed by MS2 as well as differential-mobility spectrometry combined with ozone-induced dissociation MS, we identified the carbon-carbon double bond in native 1-deoxySO to be located at the (Δ14) position. Comparing the chromatographic behavior of native 1-deoxySO to chemically synthesized SPH m18:1(14Z) and (14E) stereoisomers assigned the native compound to be SPH m18:1(14Z). This indicates that 1-deoxySLs are metabolized differently than canonical sphingolipids.

Characterisation of sphingolipids in the human lens by thin layer chromatography-desorption electrospray ionisation mass spectrometry.

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation-mass spectrometry (DESI-MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.

Radical Formation in the Gas‐Phase Ozonolysis of Deprotonated Cysteine

Although the deleterious effects of ozone on the human respiratory system are well-known, many of the precise chemical mechanisms that both cause damage and afford protection in the pulmonary epithelial lining fluid are poorly understood. As a key first step to elucidating the intrinsic reactivity of ozone with proteins, its reactions with deprotonated cysteine [Cys-H](-) are examined in the gas phase. Reaction proceeds at near the collision limit to give a rich set of products including 1) sequential oxygen atom abstraction reactions to yield cysteine sulfenate, sulfinate and sulfonate anions, and significantly 2) sulfenate radical anions formed by ejection of a hydroperoxy radical. The free-radical pathway occurs only when both thiol and carboxylate moieties are available, implicating electron-transfer as a key step in this reaction. This novel and facile reaction is also observed in small cys-containing peptides indicating a possible role for this chemistry in protein ozonolysis.

Differential-Mobility Spectrometry of 1-Deoxysphingosine Isomers: New Insights into the Gas Phase Structures of Ionized Lipids

Separation and structural identification of lipids remain a major challenge for contemporary lipidomics. Regioisomeric lipids differing only in position(s) of unsaturation are often not differentiated by conventional liquid chromatography-mass spectrometry approaches leading to the incomplete, or sometimes incorrect, assignation of molecular structure. Here we describe an investigation of the gas phase separations by differential-mobility spectrometry (DMS) of a series of synthetic analogues of the recently described 1-deoxysphingosine. The dependence of the DMS behavior on the position of the carbon-carbon double bond within the ionized lipid is systematically explored and compared to trends from complementary investigations, including collision cross-sections measured by drift tube ion mobility, reaction efficiency with ozone, and molecular dynamics simulations. Consistent trends across these modes of interrogation point to the importance of direct, through-space interactions between the charge site and the carbon-carbon double bond. Differences in the geometry and energetics of this intramolecular interaction underpin DMS separations and influence reactivity trends between regioisomers. Importantly, the disruption and reformation of these intramolecular solvation interactions during DMS are proposed to be the causative factor in the observed separations of ionized lipids which are shown to have otherwise identical collision cross-sections. These findings provide key insights into the strengths and limitations of current ion-mobility technologies for lipid isomer separations and can thus guide a more systematic approach to improved analytical separations in lipidomics.