Where You Protonate Matters: Deciphering the Unimolecular Chemistry of Protonated Myrcene and Linalool.

The unimolecular reactions of protonated myrcene and linalool were investigated by collision-induced dissociation and density functional theory calculations. Experiments on a triple quadrupole mass spectrometer showed that protonated myrcene undergoes two major unimolecular reactions losing propene and isobutene, and two minor reactions of ethene and propane loss. In each case, the product ion consists of a substituted five-member ring. Protonation of myrcene was found to form four distinct protomers, three of which can be significantly populated in the ion source. The observed fragmentation reactions were calculated and found to depend on the starting protomer. Each pathway consisted of several hydrogen-migration and ring-forming/opening steps on the way to the observed products. Likewise, protonation of linalool also produces three distinct protomers, with the global minimum being formed by protonation of a central double bond. The major reaction is water loss to form protonated myrcene, but two minor channels were also observed resulting in loss of acetone and isobutene. The calculated minimum energy reaction pathways were found to be consistent with the relative abundances of the ions in the experimental breakdown diagrams.

Determination of double bond positions in unsaturated fatty acids by pre-column derivatization with dimethyl and dipyridyl disulfide followed by LC-SWATH-MS analysis.

Comprehensive in-depth structural characterization of free mono-unsaturated and polyunsaturated fatty acids often requires the determination of carbon-carbon double bond positions due to their impact on physiological properties and relevance in biological samples or during impurity profiling of pharmaceuticals. In this research, we report on the evaluation of disulfides as suitable derivatization reagents for the determination of carbon-carbon double bond positions of unsaturated free fatty acids by UHPLC-ESI-QTOF-MS/MS analysis and SWATH (sequential windowed acquisition of all theoretical mass spectra) acquisition. Iodine-catalyzed derivatization of C = C double bonds with dimethyl disulfide (DMDS) enabled detection of characteristic carboxy-terminal MS2 fragments for various fatty acids in ESI negative mode. The determination of double bond positions of fatty acids with up to three double bonds, the transfer of the method to plasma samples, and its limitations have been shown. To achieve charge-switching for positive ion mode MS-detection, derivatization with 2,2'-dipyridyldisulfide (DPDS) was investigated. It enabled detection of both corresponding characteristic omega-end- and carboxy-end-fragments for fatty acids with up to two double bonds in positive ion mode. It provides a straightforward strategy for designing MRM transitions for targeted LC-MS/MS assays. Both derivatization techniques represent a simple and inexpensive way for the determination of double bond positions in fatty acids with low number of double bonds. No adaptation of MS hardware is required and the specific isotopic pattern of resulting sulfur-containing products provides additional structural confirmation. This reaction scheme opens up the avenue of structural tuning of disulfide reagents beyond DMDS and DPDS using reagents like cystine and analogs to achieve enhanced performance and sensitivity.

Top-down mass spectrometry analysis of capsid proteins of recombinant adeno-associated virus using multiple ion activations and proton transfer charge reduction.

Adeno-associated viruses (AAVs) are common vectors for emerging gene therapies due to their lack of pathogenicity in humans. Here, we present our investigation of the viral proteins (i.e., VP1, VP2, and VP3) of the capsid of AAVs via top-down mass spectrometry (MS). These proteins, ranging from 59 to 81 kDa, were chromatographically separated using hydrophilic interaction liquid chromatography and characterized in the gas-phase by high-resolution Orbitrap Fourier transform MS. Complementary ion dissociation methods were utilized to improve the overall sequence coverage. By reducing the overlap of product ion signals via proton transfer charge reduction on the Orbitrap Ascend BioPharma Tribrid mass spectrometer, the sequence coverage of each VP was significantly increased, reaching up to ∼40% in the case of VP3. These results showcase the improvements in the sequencing of proteins >30 kDa that can be achieved by manipulating product ions via gas-phase reactions to obtain easy-to-interpret fragmentation mass spectra.

Sequencing of Phosphorodiamidate Morpholino Oligomers by Hydrophilic Interaction Chromatography Coupled to Tandem Mass Spectrometry.

Sequencing of phosphorodiamidate morpholino oligomers (PMOs) by hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS) is reported. The MS/MS analysis was performed using a quadrupole/time-of-flight (Q-ToF) mass analyzer and collision induced dissociation (CID) in negative ion mode. To improve MS sensitivity in negative ion mode, HILIC conditions, including the separation column, mobile phases, and MS parameters, were optimized. Using the developed HILIC-CID-MS/MS method, 100% sequence coverage was achieved for PMOs ranging from 18-mer to 25-mer. Additionally, the method was successfully applied to identifying positional isomers of n - 1 deletion impurities present in PMO drug substances.

Effect of Terminal Phosphate Groups on Collisional Dissociation of RNA Oligonucleotide Anions.

The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.

Tandem mass spectrometry of π-expanded triphenylamine and N-heterotriangulene scaffolds: Radical cation versus silver(I) adduct.

Triphenylamine (TPA) and N-heterotriangulene (N-HTA) scaffolds with up to three oligophenyl extensions are investigated by electrospray ionization (tandem) mass spectrometry (ESI-[MS/]MS). Due to their low oxidation potentials, all molecules readily form radical cations in the electrospray process. The energy-resolved collision-induced dissociation behaviour of the molecular ions is contrasted to that of the silver(I) adducts. Complexation with Ag(I) leads to the expected [1:1] and [2:1] complexes (MAg+ and M2Ag+); however, even [1:2] complexes (MAg2 2+) can be detected for molecules with two and three large π-expansions to allow stabilization of two charges. The TPA scaffolds decompose only at high collision energies through the loss of peripheral tert-butyl groups. A general mechanism for this is proposed commencing with a methyl loss and followed by the release of isobutene and butyl radical moieties. The N-HTA-based scaffolds are considerably less stable and molecular ions fragment at low collision energies. This is caused by the facile loss of methyl radicals from the dimethylmethylene-bridged triangulene core. In contrast, complexation with Ag+ leads to a dramatic stabilization. Most interestingly, dissociation eventually proceeds via the loss of neutral AgCH3, which is indicative of strong bidentate, tweezer-like bonding of Ag+ to the molecules.

Sequencing of Phosphopeptides Using a Sequential Charge Inversion Ion/Ion Reaction and Electron Capture Dissociation Workflow.

Protein phosphorylation, a common post-translational modification (PTM), is fundamental in a plethora of biological processes, most importantly in modulating cell signaling pathways. Matrix-assisted laser desorption/ionization (MALDI) coupled to tandem mass spectrometry (MS/MS) is an attractive method for phosphopeptide characterization due to its high speed, low limit of detection, and surface sampling capabilities. However, MALDI analysis of phosphopeptides is constrained by relatively low abundances in biological samples and poor relative ionization efficiencies in positive ion mode. Additionally, MALDI tends to produce singly charged ions, generally limiting the accessible MS/MS techniques that can be used for peptide sequencing. For example, collision induced dissociation (CID) is readily amendable to the analysis of singly charged ions, but results in facile loss of phosphoric acid, precluding the localization of the PTM. Electron-based dissociation methods (e.g., electron capture dissociation, ECD) are well suited for PTM localization, but require multiply charged peptide cations to avoid neutralization during ECD. Conversely, phosphopeptides are readily ionized using MALDI in negative ion mode. If the precursor ions are first formed in negative ion mode, a gas-phase charge inversion ion/ion reaction could then be used to transform the phosphopeptide anions produced via MALDI into multiply charged cations that are well-suited for ECD. Herein we demonstrate a multistep workflow combining a charge inversion ion/ion reaction that first transforms MALDI-generated phosphopeptide monoanions into multiply charged cations, and then subjects these multiply charged phosphopeptide cations to ECD for sequence determination and phosphate bond localization.

Metabolite identification of salvianolic acid A in rat using post collision-induced dissociation energy-resolved mass spectrometry.

BACKGROUND: As one of the most famous natural products, salvianolic acid A (SAA) is undergoing clinical trials for the treatments of angina pectoris and coronary heart disorders. However, the in vivo metabolites of SAA have only been tentatively identified, leading to a barrier for precise therapeutical drug monitoring. METHODS: Ultra-high performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry (UPLC-Qtof-MS/MS) was firstly employed to acquire high-resolution MS1 and MS2 spectra for all metabolites. Through paying special attention onto the features of ester bond dissociation, metabolism sites were restricted at certain regions. To further determine the metabolism site, such as the monomethylated products (M23, M25, and M26), post collision-induced dissociation energy-resolved mass spectrometry (post-CID ER-MS) was proposed through programming progressive exciting energies to the second collision chamber of hybrid triple quadrupole-linear ion trap mass spectrometry (Qtrap-MS) device. RESULTS: After SAA oral administration, 29 metabolites (M1-M29), including five, thirteen, and sixteen ones in rat plasma, urine, and feces, respectively, were detected in rats. The metabolism route was initially determined by applying well-defined mass fragmentation pathways to those HR-m/z values of precursor and fragment ions. Metabolism site was limited to SAF- or DSS-unit based on the fragmentation patterns of ester functional group. Through matching the dissociation trajectories of concerned 1st-generation fragment ions with expected decomposition product anions using post-CID ER-MS strategy, M23 and M25 were unequivocally assigned as 3'-methyl-SAA and 3''-methyl-SAA, and M26 was identified as 2-methyl-SAA or 3-methyl-SAA. Hydrolysis, methylation, glucuronidation, sulfation, and oxidation were the primary metabolism channels being responsible for the metabolites' generation. CONCLUSION: Together, the metabolism regions and sites of SAA metabolites were sequentially identified based on the ester bond dissociation features and post-CID ER-MS strategy. Importantly, the present study provided a promising way to elevate the structural identification confidence of natural products and metabolites.

Functional Group Analysis of α-Pinene Oxidation Products Using Derivatization Reactions and High-Resolution Electrospray Ionization Collision-Induced Dissociation Mass Spectrometry.

Oxidation products of monoterpenes (C10H16) play a significant role as precursors for secondary organic aerosol formation. They contain several structural isomers with multifunctional groups. However, only a few of these isomers have been identified experimentally. We describe a measurement technique for identifying oxygen-containing functional groups (carbonyl, carboxyl, and hydroxyl groups) included in monoterpene oxidation products. This technique involves (i) three derivatization reactions (oximation of carbonyls by O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, methyl esterification of carboxylic acids by trimethylsilyl diazomethane, and acylation of alcohols by acetic anhydride), (ii) no preseparation high-resolution electrospray ionization mass spectrometry, and (iii) collision-induced dissociation. This technique was applied to functional group analysis of ozonolysis products for α-pinene. Multifunctional groups of known ozonolysis products were accurately identified. Furthermore, we successfully estimated the multifunctional groups of products that have not been previously reported.

Human Cerebellum Gangliosides: A Comprehensive Analysis by Ion Mobility Tandem Mass Spectrometry.

The human cerebellum is an ultraspecialized region of the brain responsible for cognitive functions and movement coordination. The fine mechanisms through which the process of aging impacts such functions are not well understood; therefore, a rigorous exploration of this brain region at the molecular level is deemed necessary. Gangliosides, sialylated glycosphingolipids, highly and specifically expressed in the human central nervous system, represent possible molecular markers of cerebellum development and aging. In this context, for a comprehensive determination of development- and age-specific components, we have conducted here a comparative profiling and structural determination of the gangliosides expressed in fetal cerebellum in two intrauterine developmental stages and aged cerebellum by ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS). Due to the high sensitivity and efficiency of separation provided by IMS MS, no less than 551 chemically distinct species were identified, which represents 4.5 times more gangliosides than ever discovered in this brain region. The detailed assessment of fetal vs aged cerebellum gangliosidome showed marked discrepancies not only in the general number of the species expressed, but also in their sialylation patterns, the modifications of the glycan core, and the composition of the ceramides. All of these characteristics are potential markers of cerebellum development and aging. The structural analysis by collision-induced dissociation (CID) documented the occurrence of GD1b (d18:1/18:0) isomer in the fetal cerebellum in the second gestational trimester, with all probability of GQ1b (t18:1/18:0) in the near-term fetus and of GQ1b (d18:1/18:0) in aged cerebellum.

Negative Electron Transfer Collision-Induced Dissociation of G-Quadruplexes: Uncovering the Guanine Radical Anion Loss Pathway.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

Effects of Nano-Electrospray Ionization Emitter Position on Unintentional In-Source Activation of Peptide and Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) typically introduces protein ions into the gas phase through nano-electrospray ionization (nESI). Many nESI setups have mobile stages for tuning the ion signal and extent of co-solute and salt adduction. However, tuning the position of the emitter capillary in nESI can have unintended downstream consequences for collision-induced unfolding or collision-induced dissociation (CIU/D) experiments. Here, we show that relatively small variations in the nESI emitter position can shift the midpoint (commonly called the "CID50" or "CIU50") potential of CID breakdown curves and CIU transitions by as much as 8 V on commercial instruments. A spatial "map" of the shift in CID50 for the loss of heme from holomyoglobin onto the emitter position on a Waters Synapt G2-Si mass spectrometer shows that emitter positions closer to the instrument inlet can result in significantly greater in-source activation, whereas different effects are found on an Agilent 6545XT instrument for the ions studied. A similar effect is observed for CID of the singly protonated leucine enkephalin peptide and Shiga toxin 1 subunit B homopentamer on the Waters Synapt G2-Si instrument. In-source activation effects on a Waters Synapt G2-Si are also investigated by examining the RMSD between CIU fingerprints acquired at different emitter positions and the shifts in CIU50 for structural transitions of bovine serum albumin and NIST monoclonal antibody.

Ion mobility-tandem mass spectrometry of bulky tert-butyl thiol ligated gold nanoparticles.

Gold nanoparticles (AuNPs) synthesized in the 1-3 nm range have a specific number of gold core atoms and outer protecting ligands. They have become one of the "hot topics" in recent decades because of their interesting physical and chemical properties. The characterization of their structures is usually achieved by crystal X-ray diffraction although the structures of some AuNPs remain unknown because they have not been successfully crystallized. An alternative method for studying the structure of AuNPs is electrospray ionization-ion mobility-tandem mass spectrometry (ESI-IM-MSMS). This research evaluated how effectively ESI-IM-MSMS using the commercially available Waters Synapt XS instrument yielded useful structural information from two AuNPs; Au23 (S-tBu)16 and Au30 (S-tBu)18 . The study used the maximum range of available collision energies along with ion mobility separation to measure the energy-dependence of the product ions and their drift times which is a measure of their spatial size. For Au23 (S-tBu)16 , the dissociation gave the masses of the outer protecting monomeric [RS-Au-SR] and trimeric [SR-Au-SR-Au-SR-Au-SR] staples where R = tBu, and complete dissociation of the outer layer Au and tBu groups to reveal the Au15 S8 core. For Au30 (S-tBu)18 , the dissociation products was primarily through the loss of the partial ligands S-tBu and tBu from the outer protecting layer and the loss of single Au4 (S-tBu)4 unit. These results showed the that ESI-IM-MSMS analysis of the smaller Au23 (S-tBu)16 gave information on all it major structural components whereas for Au30 (S-tBu)18 , the overall structural information was limited to the ligands of the outer layer.

Does deprotonated benzoic acid lose carbon monoxide in collision-induced dissociation?

Decarboxylation is known to be the major fragmentation pathway for the deprotonated carboxylic acids in collision-induced dissociation (CID). However, in the CID mass spectrum of deprotonated benzoic acid (m/z 121) recorded on a Q-orbitrap mass spectrometer, the dominant peak was found to be m/z 93 instead of the anticipated m/z 77. Based on theoretical calculations, 18 O-isotope labeling and MS3 experiments, we demonstrated that the fragmentation of benzoate anion begins with decarboxylation, but the initial phenide anion (m/z 77) can react with trace O2 in the mass analyzer to produce phenolate anion (m/z 93) and other oxygen-containing ions. Thus oxygen adducts should be considered when annotating the MS/MS spectra of benzoic acids.

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers.

Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

Mass spectrometry-based gas phase intramolecular benzyl migration in sparsentan, a novel endothelin and angiotensin II receptor antagonist.

We report a collision-induced dissociation (CID) based gas phase rearrangement study using quadrupole time-of-flight mass spectrometry coupled with liquid chromatography on a novel endothelin and angiotensin II receptor antagonist, sparsentan. We performed tandem mass spectrometry to identify precursor and fragment ion relationships and assigned structures for major fragment ions. We propose a benzyl migration mechanism based on bond length measurements in density functional theory (B3LYP/6-31+G*) optimized geometries of protonated sparsentan and its m/z 547 fragment. Protonated sparsentan undergoes loss of ethanol, which yields a resonance-stabilized benzylic cation with m/z 547, which further fragments into m/z 353 via benzyl migration, where the benzylic cation migrates to one of the nucleophilic nitrogen atoms followed by proton transfer from the sulfonamide nitrogen to a carbonyl oxygen, resulting in a neutral loss of mass 194. Further fragmentation of m/z 353 results in m/z 258, which undergoes radical and neutral loss to yield m/z 193 and 194, respectively. The proposed mechanism of generation of m/z 353 was confirmed by CID of deuterated sparsentan. Considering the importance of gas phase rearrangements of organic molecules in structural identifications as well as the novelty of the molecule, these findings will be helpful for future studies to predict gas phase benzyl migration in sparsentan analogs and for degradation product and metabolite identification of sparsentan and its analogs using LC-MS.

Ultraviolet photodissociation and collision-induced dissociation for qualitative/quantitative analysis of low molecular weight compounds by liquid chromatography-mass spectrometry.

Collision-induced dissociation (CID) is the most wildly used fragmentation technique for qualitative and quantitative determination of low molecular weight compounds (LMWC). Ultraviolet photodissociation (UVPD) has been mainly investigated for the analysis of peptides and lipids while only in a limited way for LMWC. A triple quadrupole linear ion trap instrument has been modified to allow ultraviolet photodissociation (UVPD) in the end of the q2 region enabling various workflows with and without data-dependent acquisition (DDA) combining CID and UVPD in the same LC-MS analysis. The performance of UVPD, with a 266-nm laser, is compared to CID for a mix of 90 molecules from different classes of LMWC including peptides, pesticides, pharmaceuticals, metabolites, and drugs of abuse. These two activation methods offer complementary fragments as well as common fragments with similar sensitivities for most analytes investigated. The versatility of UVPD and CID is also demonstrated for quantitative analysis in human plasma of bosentan and its desmethyl metabolite, used as model analytes. Different background signals are observed for both fragmentation methods as well as unique fragments which opens the possibility of developing a selective quantitative assay with improved sample throughput, in particular for analytes present in different matrices.

Rapid analysis of pharmaceutical and personal care products by soft microwave-based plasma ionization-linear ion trap mass spectrometer (SMPI-LTQ) in natural water.

In this study, a soft microwave plasma torch (SMPT) combined with a mass spectrometer (MS) was used for the first time as an analytical method to detect and analyze various pharmaceutical and personal care products (PPCPs) in aquatic environments without the need for sample pretreatment. For this purpose, ambient SMPT was used to generate plasma for ionizing the analyte molecules. Accordingly, nine PPCPs were identified by the SMPT-MS, and their identification was verified by collision-induced dissociation (CID). The technique's performance was verified with known PPCP samples, and the limits of detection (LOD) and quantification (LOQ) obtained over a linear range of 50-1 µg/L were 1.56 to 2.81 and 2.07 to 3.62 µg/L, respectively, with the standard addition recovery rate falling between 87.14 and 115.16%. These results show that the method has excellent sensitivity and selectivity, suggesting that SMPT can rapidly and directly detect PPCPs in environmental water, making it a promising method for rapid water quality inspection.

Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.

Concealed by darkness: Combination of NMR and HRMS reveal the molecular nature of dissolved organic matter in fractured-rock groundwater and connected surface waters.

Detailed molecular composition of solid phase extracted dissolved organic matter (SPEDOM) collected from fractured-rock groundwater was compared to connected surface river water at two different watersheds in the unconfined chalk aquifer of Champagne in France using full scan ultrahigh resolution electrospray and photoionization Fourier transform ion cyclotron mass spectrometry (FT-ICR MS), Orbitrap tandem MS (MS/MS) and 1H magnetic resonance spectroscopy (NMR). 1H NMR spectroscopy indicated that groundwater SPEDOM carried a higher contribution of aliphatic compounds while surface river waters SPEDOM were enriched in carboxyl-rich alicyclic molecules (CRAM), acetate derivatives and oxygenated units. Furthermore, we show here that use of photoionization (APPI(+)) in aquifer studies is key, ionizing about eight times more compounds than ESI in surface river water samples, specifically targeting the dissolved organic nitrogen pool, accounting for more than 50% of the total molecular space, as well as a non-polar, more aromatic fraction; with little overlap with compounds detected by ESI(-) FT-ICR MS. On the other hand, groundwater SPEDOM samples did not show similar selectivity as less molecular diversity was observed in APPI compared to ESI. Mass-difference transformation networks (MDiNs) applied to ESI(-) and APPI(+) FT-ICR MS datasets provided an overview of the biogeochemical relationships within the aquifer, revealing chemical diversity and microbial/abiotic reactions. Finally, the combination of ESI(-) FT-ICR MS and detailed Orbitrap MS/MS analysis revealed a pool of polar, anthropogenic sulfur-containing surfactants in the groundwaters, likely originating from agricultural runoff. Overall, our study shows that in this aquifer, groundwater SPEDOM contains a significantly reduced pool of organic compounds compared to surface river waters, possibly related to a combination of lack of sunlight and adsorption of high O/C formulas to mineral surfaces.