Characterization of Antigenic Oligosaccharides from Gram-Negative Bacteria via Activated Electron Photodetachment Mass Spectrometry.

Lipooligosaccharides (LOS), composed of hydrophilic oligosaccharides and hydrophobic lipid A domains, are found on the outer membranes of Gram-negative bacteria. Here we report the characterization of deacylated LOS of LPS by activated-electron photodetachment mass spectrometry. Collision induced dissociation (CID) of these phosphorylated oligosaccharides produces simple MS/MS spectra with most fragment ions arising from cleavages near the reducing end of the molecule where the phosphate groups are located. In contrast, 193 nm ultraviolet photodissociation (UVPD) generates a wide array of product ions throughout the oligosaccharide including cross-ring fragments that illuminate the branching patterns. However, there are also product ions that are redundant or uninformative, resulting in more congested spectra that complicate interpretation. In this work, a hybrid UVPD-CID approach known as activated-electron photodetachment (a-EPD) affords less congested spectra than UVPD alone and richer fragmentation patterns than CID alone. a-EPD combines UVPD of negatively charged oligosaccharides to yield abundant charge-reduced radical ions which are subsequently interrogated by collisional activation. CID of the charge-reduced precursors results in extensive fragmentation throughout the backbone of the oligosaccharide. This hybridized a-EPD approach was employed to characterize the structure and branching pattern of deacylated LOS of E. coli.

A mixed methods exploratory study tackling smoking during pregnancy in an urban Aboriginal and Torres Strait Islander primary health care service.

BACKGROUND: Pregnancy can be a time of joy and a time of significant stress. For many Aboriginal and Torres Strait Islander (hereafter, respectfully, Indigenous) women, cigarette smoking, even during pregnancy, is a socially sanctioned behavioural response to stress. Indigenous women smoke during pregnancy at higher rates than their non-Indigenous counterparts. METHODS: A mixed methods, exploratory study, undertaken in an urban, Indigenous primary health care service, tested the impact and acceptability of a smoking cessation intervention for women pregnant with an Indigenous baby, their significant other (SO), and their primary health care service. The intervention included case management, incentivised smoking cessation support and culturally-based art activities. RESULTS: Thirty-one pregnant women and 16 SOs participated. Nearly half attempted to quit at least once during the study, 36% (4/11) of pregnant women had quit at the 3 month assessment and two remained smoke free 1 month postpartum. Most participants self-reported a reduction in tobacco smoking. Exhaled CO confirmed this for SOs (mean reduction - 2.2 ppm/assessment wave, 95% CI: -4.0, - 0.4 ppm/assessment wave, p = 0.015) but not for pregnant women. Many participants experienced social and economic vulnerabilities, including housing and financial insecurity and physical safety concerns. CONCLUSIONS: Tobacco smoking is normalised and socially sanctioned in Indigenous communities and smoking is frequently a response to the multitude of stressors and challenges that Indigenous people experience on a daily basis. Smoking cessation interventions for pregnant Indigenous women must be cognisant of the realities of their private lives where the smoking occurs, in addition to the impact of the broader societal context. Narrow definitions of success focussing only on smoking cessation ignore the psychological benefit of empowering women and facilitating positive changes in smoking behaviours. Our smoking cessation intervention supported pregnant women and their SOs to manage these stressors and challenges, thereby enabling them to develop a solid foundation from which they could address their smoking. A broad definition of success in this space is required: one that celebrates positive smoking behaviour changes in addition to cessation.

Mapping phosphate modifications of substituted lipid A via a targeted MS3 CID/UVPD strategy.

Structural characterization of lipid A from Gram-negative bacteria remains a significant challenge, especially with respect to localizing modifications of the phosphate groups typically found on the reducing and non-reducing ends of the ß-1',6-linked glucosamine disaccharide backbone of lipid A. As reported here, combining traditional collisional activated dissociation (CAD) and ultraviolet photodissociation (UVPD) in a hybrid MS3 approach facilitates identification and localization of substituents of the phosphate groups. The focus is on rapid identification and characterization of substituted lipid A species with specific emphasis on the modifications on the 1 and 4' phosphate moieties. Mapping these modifications, typically ones that modify the surface charges of lipopolysaccharides, is particularly important owing to the impact of these types of modifications on antibiotic resistance. The presence of phosphoethanolamine, aminoarabinose, and galactosamine moieties in hexaacylated and heptaacylated lipid A species, including ones from Enterobacter cloacae and Acinetobacter baumannii, are characterized using a targeted MS3 strategy to identify glycosidic product ions (1,5X1 and 0,4A2, typically) which allow localization of the substituents.

Comparing Positively and Negatively Charged Distonic Radical Ions in Phenylperoxyl Forming Reactions.

In the gas phase, arylperoxyl forming reactions play a significant role in low-temperature combustion and atmospheric processing of volatile organic compounds. We have previously demonstrated the application of charge-tagged phenyl radicals to explore the outcomes of these reactions using ion trap mass spectrometry. Here, we present a side-by-side comparison of rates and product distributions from the reaction of positively and negatively charge tagged phenyl radicals with dioxygen. The negatively charged distonic radical ions are found to react with significantly greater efficiency than their positively charged analogues. The product distributions of the anion reactions favor products of phenylperoxyl radical decomposition (e.g., phenoxyl radicals and cyclopentadienone), while the comparable fixed-charge cations yield the stabilized phenylperoxyl radical. Electronic structure calculations rationalize these differences as arising from the influence of the charged moiety on the energetics of rate-determining transition states and reaction intermediates within the phenylperoxyl reaction manifold and predict that this influence could extend to intra-molecular charge-radical separations of up to 14.5 Å. Experimental observations of reactions of the novel 4-(1-carboxylatoadamantyl)phenyl radical anion confirm that the influence of the charge on both rate and product distribution can be modulated by increasing the rigidly imposed separation between charge and radical sites. These findings provide a generalizable framework for predicting the influence of charged groups on polarizable radicals in gas phase distonic radical ions. Graphical Abstract.

Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry.

Complete structural characterization of complex lipids, such as glycerophospholipids, by tandem mass spectrometry (MS/MS) continues to present a major challenge. Conventional activation methods do not generate fragmentation patterns that permit the simultaneous discernment of isomers which differ in both the positions of acyl chains on the glycerol backbone and the double bonds within the acyl chains. Herein we describe a hybrid collisional activation/UVPD workflow that yields near-complete structural information for glycerophospholipids. This hybrid MS3 strategy affords the lipid's sum composition based on the accurate mass measured for the intact lipid as well as highly specific diagnostic product ions that reveal both the acyl chain assignment (i.e., sn-position) and the site-specific location of double bonds in the acyl chains. This approach is demonstrated to differentiate sn-positional and double-bond-positional isomers, such as the regioisomeric phosphatidylcholines PC 16:0/18:1(n-9) and PC 18:1(n-9)/16:0, and has been integrated into an LC-MS3 workflow.

Mass spectrometric identification of the N-monosubstituted N-hydroxylamino functionality in protonated analytes via ion/molecule reactions in tandem mass spectrometry.

RATIONALE: N-Monosubstituted hydroxylamines correspond to an important class of metabolites for many bioactive molecules. In this study, a tandem mass spectrometric method based on ion/molecule reactions was developed for the identification of compounds with the N-monosubstituted hydroxylamino functionality. METHODS: The diagnostic ion/molecule reaction occurs between protonated analytes with 2-methoxypropene (MOP) inside a linear quadrupole ion trap mass spectrometer. RESULTS: Most protonated compounds with N-monosubstituted and disubstituted hydroxylamino and oxime functional groups react with MOP via proton transfer and formation of a stable adduct in a linear quadrupole ion trap mass spectrometer. However, only protonated compounds with N-monosubstituted hydroxylamino groups form the characteristic MOP adduct-MeOH product. Possible mechanisms of this reaction are discussed. CONCLUSIONS: A method based on functional group-selective ion/molecule reactions in a linear quadrupole ion trap mass spectrometer has been demonstrated to allow the identification of protonated compounds with the N-monosubstituted hydroxylamino functionality. Only N-monosubstituted hydroxylamines react with MOP via formation of an adduct that has eliminated methanol.

Identification of the sulfoxide functionality in protonated analytes via ion/molecule reactions in linear quadrupole ion trap mass spectrometry.

A mass spectrometric method utilizing gas-phase ion/molecule reactions of 2-methoxypropene (MOP) has been developed for the identification of the sulfoxide functionality in protonated analytes in a LQIT mass spectrometer. Protonated sulfoxide analytes react with MOP to yield an abundant addition product (corresponding to 37-99% of the product ions), which is accompanied by a much slower proton transfer. The total efficiency (percent of gas-phase collisions leading to products) of the reaction is moderate (3-14%). A variety of compounds with different functional groups, including sulfone, hydroxylamino, N-oxide, aniline, phenol, keto, ester, amino and hydroxy, were examined to probe the selectivity of this reaction. Most of the protonated compounds with proton affinities lower than that of MOP react mainly via proton transfer to MOP. The formation of adduct-MeOH ions was found to be characteristic for secondary N-hydroxylamines. N-Oxides formed abundant MOP adducts just like sulfoxides, but sulfoxides can be differentiated from N-oxides based on their high reaction efficiencies. The reaction was tested by using the anti-inflammatory drug sulindac (a sulfoxide) and its metabolite sulindac sulfone. The presence of a sulfoxide functionality in the drug but a sulfone functionality in the metabolite was readily demonstrated. The presence of other functionalities in addition to sulfoxide in the analytes was found not to influence the diagnostic reactivity.

Identification of the sulfone functionality in protonated analytes via ion/molecule reactions in a linear quadrupole ion trap mass spectrometer.

A tandem mass spectrometric method is presented for the rapid identification of drug metabolites that contain the sulfone functional group. This method is based on a gas-phase ion/molecule reaction of protonated sulfone analytes with trimethyl borate (TMB) that yields a diagnostic product ion, adduct-Me2O, at high reaction efficiency. A variety of compounds with different functional groups, such as sulfoxides, hydroxylamines, N-oxides, anilines, phenol, an aliphatic amine, and an aliphatic alcohol, were examined to probe the selectivity of this reaction. Except for protonated sulfones, most of the protonated compounds react very slowly or not at all with TMB. Most importantly, none of them give the adduct-Me2O product. A mechanism that explains the observed selectivity is proposed for the diagnostic reaction and is supported by quantum chemical calculations. The reaction was tested with the anti-inflammatory drug sulindac and its metabolite, sulindac sulfone, which were readily distinguished. The presence of other functionalities in addition to sulfone was found not to influence the diagnostic reactivity.

UV-induced effects on chlorination of creatinine.

Ultraviolet (UV) irradiation is commonly employed for water treatment in swimming pools to complement conventional chlorination, and to reduce the concentration of inorganic chloramine compounds. The approach of combining UV irradiation and chlorination has the potential to improve water quality, as defined by microbial composition. However, relatively little is known about the effects of this process on water chemistry. To address this issue, experiments were conducted to examine the effects of sequential UV254 irradiation/chlorination, as will occur in recirculating system of swimming pools, on disinfection byproduct (DBP) formation. Creatinine, which is present in human sweat and urine, was selected as the target precursor for these experiments. Enhanced formation of dichloromethylamine (CH3NCl2) and inorganic chloramines was observed to result from post-chlorination of UV-irradiated samples. Chlorocreatinine was found to be more sensitive to UV254 irradiation than creatinine; UV254 irradiation of chlorocreatinine resulted in opening of the ring structure, thereby yielding a series of intermediates that were more susceptible to free chlorine attack than their parent compound. The quantum yields for photodegradation of creatinine and chlorocreatinine at 254 nm were estimated at 0.011 ± 0.002 mol/E and 0.144 ± 0.011 mol/E, respectively. The N-Cl bond was found to be common to UV-sensitive chlorinated compounds (e.g., inorganic chloramines, CH3NCl2, and chlorocreatinine); compounds that were less susceptible to UV-based attack generally lacked the N-Cl bond. This suggested that the N-Cl bond is susceptible to UV254 irradiation, and cleavage of the N-Cl bond appears to open or promote reaction pathways that involve free chlorine, thereby enhancing formation of some DBPs and promoting loss of free chlorine. Proposed reaction mechanisms to describe this behavior based on creatinine as a precursor are presented.

Effects of a hydroxyl substituent on the reactivity of the 2,4,6-tridehydropyridinium cation, an aromatic σ,σ,σ-triradical.

The reactivity of 3-hydroxy-2,4,6-tridehydropyridinium cation was found to be drastically different from the reactivity of 2,4,6-tridehydropyridinium cation. While the latter triradical reacts with tetrahydrofuran, dimethyl disulfide and ally iodide via three consecutive atom or group abstractions, the former triradical exhibits this behavior only with tetrahydrofuran. Only a single atom or group abstraction was observed for the 3-hydroxy-2,4,6-tridehydropyridinium cation upon interaction with dimethyl disulfide and allyl iodide. This change in reactivity is caused by the hydroxyl group that strengthens the interactions between the two radical sites adjacent to it, thus reducing their reactivity. This explanation is supported by the observation of similar behavior for related biradicals.