C. Ruth Wang

Neonicotinoids and organophosphates are important analytes due to their use as insecticides and environmental profiles. Cationized imidacloprid and chlorpyrifos as prototypical examples of these were analyzed with drift tube, traveling wave, trapped ion, and cyclic ion mobility mass spectrometry, with experimental collision cross sections (ExpCCSN2) derived for each to help understand their gas phase structures. The ExpCCSN2 of potassiated imidacloprid and potassiated chlorpyrifos are reported for the first time. The cross-platform ExpCCSN2 for [imidacloprid + K]+ is 160–165 Å2 and for [chlorpyrifos + K]+ is 175–176 Å2. Consistent assignment of the [imidacloprid + H]+ structure is achieved based on combining evidence from current work, literature interlaboratory CCS values, DFT energetics, predicted CCS (CalcCCSN2) and literature infrared multiple photon dissociation (IRMPD) spectroscopy. As expected, all cationized species here form charge solvated structures in the gas phase. Imidacloprid coordinates all three cations at the pyridinic N, with a varying degree of nitroguanidine non-covalent interaction for each, depending on the cation. Sodiated and potassiated imidacloprid ions have a 10-membered ring structure where the overall size trend depends more on the non-covalent interaction with nitroguanidine than the incorporated cation size, resulting in different structures. Chlorpyrifos also coordinates cations at the N, but consistently forms 6-membered ring structures. Sodiated and potassiated species change in size relative to the size of the incorporated cation. The consistency of interlaboratory results and current results also support confident assignment for the protomer of [chlorpyrifos + H]+.

Neonicotinoids and organophosphates are important analytes due to their use as insecticides and environmental profiles. Cationized imidacloprid and chlorpyrifos as prototypical examples of these were analyzed with drift tube, traveling wave, trapped ion, and cyclic ion mobility mass spectrometry, with experimental collision cross sections ( ^Exp CCS _N2 ) derived for each to help understand their gas phase structures. The ^Exp CCS _N2 of potassiated imidacloprid and chlorpyrifos are reported for the first time. The cross-platform ^Exp CCS _N2 for [imidacloprid+K] ⁺ is 160−165 Ų and for [chlorpyrifos+K] ⁺ is 175–176 Ų. Consistent assignment of [imidacloprid+H] ⁺ structure is achieved based on current work, literature interlaboratory CCS values, DFT energetics, predicted CCS ( ^Calc CCS _N2 ) and literature IRMPD spectroscopy. Imidacloprid coordinates cations at the pyridinic N, with a varying degree of nitroguanidine interaction that depends on the cation. Sodiated and potassiated imidacloprid ions have a 10-membered ring structure where the overall size depends more on the non-covalent interactions than the incorporated cation size. Chlorpyrifos also coordinates cations at the N, but consistently forms 6-membered ring structures. Overall size trends for sodiated and potassiated species follow the cation size trends, revealing the change in size due to incorporated cation. The consistency of interlaboratory results and current results also support confident assignment for the protomer of [chlorpyrifos+H] ⁺ .

Infections caused by the opportunistic fungal pathogen Aspergillus fumigatus pose a serious public health system burden. The inherent limitations in existing antifungal drugs in conjunction with a rising emergence of antifungal resistance emphasizes an urgent need to identify and target alternative pathways crucial to survival and virulence. Targeting the fungal mannitol biosynthesis enzymes provides a promising avenue in the development of new antifungals due to the multifaceted roles mannitol fulfils in the fungal life cycle. However, a distinct lack of available structural information for these enzymes has hindered drug discovery efforts. We report the first crystal structure of mannitol-2-dehydrogenase from A. fumigatus in an unbound monomeric state (1.8 Å) and bound to its co-factor NADH (2.1 Å), via. a large, central cavity lined with positively charged residues that readily accommodates NADH. This interaction is further stabilised by a network of hydrogen bond interactions and π-π stacking between Phe45 and the nicotinamide ring of NADH. Furthermore, rigorous kinetic characterisation of A. fumigatus mannitol-2-dehydrogenase demonstrates the dose-dependent inhibitory activity of 1,4-benzoquinone, a cysteine-modifying small molecule inhibitor (IC ₅₀ = 1.2 ± 0.2 nM). In addition, intact MS and proteomic analysis further reveal that 1,4-benzoquinone modifies up to five cysteine residues of mannitol-2-dehydrogenase and displays antifungal activity against A. fumigatus , which is enhanced in combination with a front-line antifungal voriconazole. From this work, we have established the foundations for a novel antifungal drug discovery avenue that targets the fungal mannitol biosynthesis pathway to better treat aspergillosis and related pathogenic infections.

Neonicotinoids and organophosphates are important analytes due to their use as insecticides and environmental profiles. Cationized imidacloprid and chlorpyrifos as prototypical examples of these were analyzed with drift tube, traveling wave, trapped ion, and cyclic ion mobility mass spectrometry, with experimental collision cross sections ( ^Exp CCS _N2 ) derived for each to help understand their gas phase structures. The ^Exp CCS _N2 of potassiated imidacloprid and chlorpyrifos are reported for the first time. The cross-platform ^Exp CCS _N2 for [imidacloprid+K] ⁺ is 160−165 Ų and for [chlorpyrifos+K] ⁺ is 175–176 Ų. Consistent assignment of [imidacloprid+H] ⁺ structure is achieved based on current work, literature interlaboratory CCS values, DFT energetics, predicted CCS ( ^Calc CCS _N2 ) and literature IRMPD spectroscopy. Imidacloprid coordinates cations at the pyridinic N, with a varying degree of nitroguanidine interaction that depends on the cation. Sodiated and potassiated imidacloprid ions have a 10-membered ring structure where the overall size depends more on the non-covalent interactions than the incorporated cation size. Chlorpyrifos also coordinates cations at the N, but consistently forms 6-membered ring structures. Overall size trends for sodiated and potassiated species follow the cation size trends, revealing the change in size due to incorporated cation. The consistency of interlaboratory results and current results also support confident assignment for the protomer of [chlorpyrifos+H] ⁺ .

The gas-phase cross-platform collision cross sections (CCS) of cationized (protonated, sodiated, and potassiated) imidacloprid and chlorpyrifos were analyzed with single- and multi-field drift tube, traveling wave, trapped ion, and cyclic ion mobility mass spectrometry. The experimental cross-platform CCS are in the range of: [imidacloprid+H]+ = 154−158 Å; [imidacloprid+Na]+ = 158–160 Ų; [imidacloprid+K]+ = 160−165 Ų; [chlorpyrifos+H]+ = 163−166 Ų; [chlorpyrifos+Na]+ = 170−173 Ų, and [chlorpyrifos+K]+ = 175–176 Ų, and are within ~3% of each other and inter-laboratory values. DFT predicts imidacloprid coordinates cations at the pyridinic N, with a degree of nitroguanidine interaction that depends on the cation, while chlorpyrifos also coordinates cations at the N, but consistently forms 6-membered ring structures. The theoretically predicted CCS of protonated imidacloprid deviated significantly (7−10%) from the experimental CCS. In contrast, sodiated and potassiated imidacloprid ions’ theoretically predicted CCS were consistent (2−3%) with the experimental CCS, supporting a more compact 9-membered ring-shaped structure compared to the protonated congener. On the other hand, predicted CCS for protonated chlorpyrifos matched the experimental CCS well (2−5%), but sodiated and potassiated chlorpyrifos had less agreement between calculated and experimental CCS (>5.5%), however structural trends are reproduced. While cross-platform variations are minimal, discrepancies in predicted CCS highlight the need for standardized protocols before CCS can reliably identify pesticide residues.

Snake venoms are comprised of bioactive proteins and peptides that facilitate severe snakebite envenomation symptoms. A comprehensive understanding of venom compositions and the subtle heterogeneity therein is important. While bottom-up proteomics has been the well-established approach to catalogue venom compositions, top-down proteomics has emerged as a complementary strategy to characterize venom heterogeneity at the intact protein level. However, top-down proteomics has not been as widely implemented in the snake venom field as bottom-up proteomics, with various emerging top-down methods yet to be developed for venom systems. Here, we have explored three main top-down mass spectrometry methodologies in a proof-of-concept study to characterize selected three-finger toxin and phospholipase A(2) proteoforms from the forest cobra (Naja melanoleuca) venom. We demonstrated the utility of a data-independent acquisition mode "MSE" for untargeted fragmentation on a chromatographic time scale and its improvement in protein sequence coverage compared to conventional targeted tandem mass spectrometry analysis. We also showed that protein identification can be further improved using a hybrid fragmentation approach, combining electron-capture dissociation and collision-induced dissociation. Lastly, we reported the promising application of multifunctional cyclic ion mobility separation and post-ion mobility fragmentation on snake venom proteins for the first time.

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe–N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe–N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.