Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5'-Deoxyadenosyl Radical.

Radical S-adenosyl-l-methionine (SAM) enzymes employ a [4Fe-4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with g∥ = 2.035 and g⊥ = 2.004 observed when the reaction is freeze-quenched at 500 ms. Thermal annealing of frozen Ω converts it into a second paramagnetic species centered at giso = 2.004; this second species was generated directly using freeze-quench at intermediate times (∼8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy as the tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed in samples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5'-deoxyadenosyl radical (5'-dAdo•). The time course of the 5'-dAdo• and tertiary peptide radical EPR signals reveals that the former generates the latter. These results thus support a mechanism in which Ω liberates 5'-dAdo• by Fe-C5' bond homolysis, and the 5'-dAdo• attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. The results thus provide a picture of a catalytically competent 5'-dAdo• intermediate trapped just prior to reaction with the substrate.

Online Coupling of Liquid Chromatography with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at 21 T Provides Fast and Unique Insight into Crude Oil Composition.

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry provides the highest mass resolving power and mass measurement accuracy for detailed characterization of complex chemical mixtures. Here, we report the coupling of online liquid chromatography of complex mixtures with a 21 tesla FT-ICR mass spectrometer. The high magnetic field enables large ion populations to be analyzed for each spectrum for a high dynamic range, with 3.2 million mass resolving power at m/z 400 (6.2 s transient duration) or 1.6 million (3.1 s transient duration) while maintaining high mass accuracy for molecular formula assignment (root-mean-square assignment error < 0.150 ppm). Thousands of unique elemental compositions are assigned per mass spectrum, which can be grouped by the heteroatom class, double bond equivalents (the number of rings and double bonds to carbon), and carbon number. Figures of merit are discussed, as well as characterization of an Arabian heavy vacuum gas oil in terms of the ring number, compound class, double bond equivalents, and ion type. Consideration of elemental composition and retention order provides additional structural information.

Hydrocarbons to carboxyl-rich alicyclic molecules: A continuum model to describe biodegradation of petroleum-derived dissolved organic matter in contaminated groundwater plumes.

Relationships between dissolved organic matter (DOM) reactivity and chemical composition in a groundwater plume containing petroleum-derived DOM (DOMHC) were examined by quantitative and qualitative measurements to determine the source and chemical composition of the compounds that persist downgradient. Samples were collected from a transect down the core of the plume in the direction of groundwater flow. An exponential decrease in dissolved organic carbon concentration resulting from biodegradation along the transect correlated with a continuous shift in fluorescent DOMHC from shorter to longer wavelengths. Moreover, ultrahigh resolution mass spectrometry showed a shift from low molecular weight (MW) aliphatic, reduced compounds to high MW, unsaturated (alicyclic/aromatic), high oxygen compounds that are consistent with carboxyl-rich alicyclic molecules. The degree of condensed aromaticity increased downgradient, indicating that compounds with larger, conjugated aromatic core structures were less susceptible to biodegradation. Nuclear magnetic resonance spectroscopy showed a decrease in alkyl (particularly methyl) and an increase in aromatic/olefinic structural motifs. Collectively, data obtained from the combination of these complementary analytical techniques indicated that changes in the DOMHC composition of a groundwater plume are gradual, as relatively low molecular weight (MW), reduced, aliphatic compounds from the oil source were selectively degraded and high MW, alicyclic/aromatic, oxidized compounds persisted.

Barium ion adduct mass spectrometry to identify carboxylic acid photoproducts from crude oil-water systems under solar irradiation.

Petroleum derived dissolved organic matter (DOMHC) samples were successfully cationized with barium, revealing many [M-H + Ba]+ peaks in both dark and simulated sunlight treatments. The DOMHC samples generated after light exposure exhibited a greater number of [M-H + Ba]+ peaks compared to the dark control. Multiple [M-H + Ba]+ peaks were investigated in the irradiated DOMHC using low resolution MS/MS in order to confirm the presence of diagnostic fragment ions, m/z 139, 155 and 196 in each treatment. Due to the high complexity of the bariated DOMHC mixture, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS/MS) was employed to obtain molecular level information for both irradiated and dark treatments. The irradiated DOMHC treatments had more bariated oxygenated species over a wide range of H/C and O/C when compared to the dark controls. Doubly bariated species were also observed in DOMHC, which provides evidence that photochemistry transforms DOMHC to even more complex mixtures with multiple oxygenations per molecule. This study provides evidence that barium adduct mass spectrometry can be successfully applied to DOMHC screening for the presence of COOHs, both in dark samples and solar irradiated samples. Furthermore, direct evidence and molecular composition of aqueous phase crude oil photoproducts is provided by this technique.

Ultrahigh Resolution Ion Isolation by Stored Waveform Inverse Fourier Transform 21 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Stored waveform inverse Fourier transform (SWIFT) is a versatile method to generate complex isolation/ejection waveforms for precursor isolation prior to tandem mass spectrometry experiments. Here, we report ultrahigh resolving power ion isolation by SWIFT on a 21 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Individual histone proteoforms are isolated (0.6 m/z isolation window) with near 100% efficiency using a 52 ms SWIFT isolation, followed by in-cell fragmentation by ultraviolet photodissociation (UVPD). Ion isolation resolving power of 175 000 (m/Δm) is demonstrated by isolation of individual peaks at a spacing of 0.0034 Da at m/z 597 from a complex mixture of Canadian bitumen. An individual m/z ion, which corresponds to a single elemental composition, from a complex mixture is isolated and fragmented by infrared multiphoton dissociation (IRMPD). Theoretical and experimental considerations that limit achievable ion isolation resolving power are discussed.

Ultra-High Mass Resolving Power, Mass Accuracy, and Dynamic Range MALDI Mass Spectrometry Imaging by 21-T FT-ICR MS.

Detailed characterization of complex biological surfaces by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) requires instrumentation that is capable of high mass resolving power, mass accuracy, and dynamic range. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers the highest mass spectral performance for MALDI MSI experiments, and often reveals molecular features that are unresolved on lower performance instrumentation. Higher magnetic field strength improves all performance characteristics of FT-ICR; mass resolving power improves linearly, while mass accuracy and dynamic range improve quadratically with magnetic field strength. Here, MALDI MSI at 21T is demonstrated for the first time: mass resolving power in excess of 1 600 000 (at m/z 400), root-mean-square mass measurement accuracy below 100 ppb, and dynamic range per pixel over 500:1 were obtained from the direct analysis of biological tissue sections. Molecular features with m/z differences as small as 1.79 mDa were resolved and identified with high mass accuracy. These features allow for the separation and identification of lipids to the underlying structures of tissues. The unique molecular detail, accuracy, sensitivity, and dynamic range combined in a 21T MALDI FT-ICR MSI experiment enable researchers to visualize molecular structures in complex tissues that have remained hidden until now. The instrument described allows for future innovative, such as high-end studies to unravel the complexity of biological, geological, and engineered organic material surfaces with an unsurpassed detail.

Correction to: Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

In the preceding article "Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate" by Oyler et al., an error in the J5 E. coli LPS chemical structure (Figs. 2 and 4) was introduced and propagated into the final revision.

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract ᅟ.

21 Tesla FT-ICR Mass Spectrometer for Ultrahigh-Resolution Analysis of Complex Organic Mixtures.

We describe complex organic mixture analysis by 21 tesla (T) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh mass-resolving power (m/Δm50% > 2 700 000 at m/z 400) and mass accuracy (80 ppb rms) enable resolution and confident identification of tens of thousands of unique elemental compositions. We demonstrate 2.2-fold higher mass-resolving power, 2.6-fold better mass measurement accuracy, and 1.3-fold more assigned molecular formulas compared to our custom-built, state-of-the-art 9.4 T FT-ICR mass spectrometer for petroleum and dissolved organic matter (DOM) analyses. Analysis of a heavy petroleum distillate exemplifies the need for ultrahigh-performance mass spectrometry (49 040 assigned molecular formulas for 21 T versus 29 012 for 9.4 T) and extends the identification of previously unresolved Oo, SsOo, and NOo classes. Mass selective ion accumulation (20 Thompson isolation) of an asphalt volcano sample yields 462 resolved mass spectral peaks at m/z 677 and reveals previously unresolved CcHhNnOoSs mass differences at high mass (m/z > 600). Similar performance gains are realized in the analysis of dissolved organic matter, where doubly charged Oo species are resolved from singly charged SOo species, which requires a mass-resolving power greater than 1 400 000 (at m/z 600). This direct comparison reveals the continued need for higher mass-resolving power and better mass accuracy for comprehensive molecular characterization of the most complex organic mixtures.

Combining Time-of-Flight Secondary Ion Mass Spectrometry Imaging Mass Spectrometry and CARS Microspectroscopy Reveals Lipid Patterns Reminiscent of Gene Expression Patterns in the Wing Imaginal Disc of Drosophila melanogaster.

Using label-free ToF-SIMS imaging mass spectrometry, we generated a map of small molecules differentially expressed in the Drosophila wing imaginal disc. The distributions of these moieties were in line with gene expression patterns observed during wing imaginal disc development. Combining ToF-SIMS imaging and coherent anti-Stokes Raman spectroscopy (CARS) microspectroscopy allowed us to locally identify acylglycerols as the main constituents of the pattern differentiating the future body wall tissue from the wing blade tissue. The findings presented herein clearly demonstrate that lipid localization patterns are strongly correlated with a developmental gene expression. From this correlation, we hypothesize that lipids play a so far unrecognized role in organ development.

Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry.

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.

Changing the Face of Veterinary Medicine: Research and Clinical Developments at AAVMC Institutions.

This paper provides a 50-year overview of research and clinical advances in AAVMC member colleges in four representative fields of veterinary medicine: oncology, vaccine development, production medicine, and public health. Though emphasis is on the progress since the mid-1960s, the salient background and associated personnel in each field are also identified to the extent that their description informs more recent events. Advances in board certification and post-graduate clinical and research educational opportunities are also described.

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis.

We describe the design and initial performance of the first 21 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The 21 tesla magnet is the highest field superconducting magnet ever used for FT-ICR and features high spatial homogeneity, high temporal stability, and negligible liquid helium consumption. The instrument includes a commercial dual linear quadrupole trap front end that features high sensitivity, precise control of trapped ion number, and collisional and electron transfer dissociation. A third linear quadrupole trap offers high ion capacity and ejection efficiency, and rf quadrupole ion injection optics deliver ions to a novel dynamically harmonized ICR cell. Mass resolving power of 150,000 (m/Δm(50%)) is achieved for bovine serum albumin (66 kDa) for a 0.38 s detection period, and greater than 2,000,000 resolving power is achieved for a 12 s detection period. Externally calibrated broadband mass measurement accuracy is typically less than 150 ppb rms, with resolving power greater than 300,000 at m/z 400 for a 0.76 s detection period. Combined analysis of electron transfer and collisional dissociation spectra results in 68% sequence coverage for carbonic anhydrase. The instrument is part of the NSF High-Field FT-ICR User Facility and is available free of charge to qualified users.

Mass spectrometry imaging of biological tissue: an approach for multicenter studies.

Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDI-Fourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common data format (imzML) and a public data repository can contribute to more reliability and transparency of MS imaging studies.

Distributed computing strategies for processing of FT-ICR MS imaging datasets for continuous mode data visualization.

High-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging enables the spatial mapping and identification of biomolecules from complex surfaces. The need for long time-domain transients, and thus large raw file sizes, results in a large amount of raw data ("big data") that must be processed efficiently and rapidly. This can be compounded by large-area imaging and/or high spatial resolution imaging. For FT-ICR, data processing and data reduction must not compromise the high mass resolution afforded by the mass spectrometer. The continuous mode "Mosaic Datacube" approach allows high mass resolution visualization (0.001 Da) of mass spectrometry imaging data, but requires additional processing as compared to feature-based processing. We describe the use of distributed computing for processing of FT-ICR MS imaging datasets with generation of continuous mode Mosaic Datacubes for high mass resolution visualization. An eight-fold improvement in processing time is demonstrated using a Dutch nationally available cloud service.

Systematic review of an emerging trend in china: resting-state functional connectivity in major depressive disorder.

Major depressive disorder continues to challenge medical and psychological resources worldwide. A marked surge has occurred recently in China in neuroimaging studies of major depressive disorder. Those studies represent an emerging trend in neuropsychiatry in that such research has previously been extremely rare in China. The present article provides a systematic review of reports published in English by research institutes in China on resting-state functional connectivity studied by MRI in depressed subjects and healthy control subjects. Particular attention is given to whether the information may advance effective diagnosis and treatment options for patients with major depressive disorder.

Exploratory meta-analysis on deep brain stimulation in treatment-resistant depression.

OBJECTIVE: Deep brain stimulation is currently an experimental treatment for major depressive disorder. Information is lacking, however, on how sham responding may affect efficacy. This article applies exploratory meta-analysis to address that topic. METHODS: Data on benefits of deep brain electrical stimulation come from a recent review. Stimulated brain regions included subgenual cingulate, capsular interna, nucleus accumbens, and medial forebrain bundle. Expert opinion plus random number software was used to generate hypothetical values for sham responding. RESULTS: An effect size of 1.71 (95% CI: 1.47-1.96) was obtained for deep brain stimulation versus sham treatment in patients suffering from long-term treatment-resistant depression. CONCLUSION: Preliminary findings on deep brain electrical stimulation suggest that the procedure may be 71% more effective than sham treatment. Expressing these findings as patients-needed-to-treat, deep brain electrical stimulation is required by 2.9 patients with long-term treatment-resistant depression in order for one of them to benefit.

Experimental investigation of the 2D ion beam profile generated by an ESI octopole-QMS system.

In this paper, we have employed an ion imaging approach to investigate the behavior of ions exiting from a quadrupole mass spectrometer (QMS) system that employs a radio frequency octopole ion guide before the QMS. An in-vacuum active pixel detector (Timepix) is employed at the exit of the QMS to image the ion patterns. The detector assembly simultaneously records the ion impact position and number of ions per pixel in every measurement frame. The transmission characteristics of the ion beam exiting the QMS are studied using this imaging detector under different operating conditions. Experimental results confirm that the ion spatial distribution exiting the QMS is heavily influenced by ion injection conditions. Furthermore, ion images from Timepix measurements of protein standards demonstrate the capability to enhance the quality of the mass spectral information and provide a detailed insight in the spatial distribution of different charge states (and hence different m/z) ions exiting the QMS.

Secondary ion mass spectrometry imaging of Dictyostelium discoideum aggregation streams.

High resolution imaging mass spectrometry could become a valuable tool for cell and developmental biology, but both, high spatial and mass spectral resolution are needed to enable this. In this report, we employed Bi3 bombardment time-of-flight (Bi3 ToF-SIMS) and C60 bombardment Fourier transform ion cyclotron resonance secondary ion mass spectrometry (C60 FTICR-SIMS) to image Dictyostelium discoideum aggregation streams. Nearly 300 lipid species were identified from the aggregation streams. High resolution mass spectrometry imaging (FTICR-SIMS) enabled the generation of multiple molecular ion maps at the nominal mass level and provided good coverage for fatty acyls, prenol lipids, and sterol lipids. The comparison of Bi3 ToF-SIMS and C60 FTICR-SIMS suggested that while the first provides fast, high spatial resolution molecular ion images, the chemical complexity of biological samples warrants the use of high resolution analyzers for accurate ion identification.

PET brain imaging of neuropeptide Y2 receptors using N-11C-methyl-JNJ-31020028 in pigs.

UNLABELLED: Neuropeptide Y2 (NPY2) receptors are implicated in diverse brain disorders, but no suitable PET radiotracers are currently available for studying NPY2 receptors in the living brain. We developed a novel positron-emitting radioligand based on the NPY2 receptor antagonist JNJ-31020028 (N-(4-(4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl)-3-fluorophenyl)-2-pyridin-3-ylbenzamide) and used the radiotracer for PET brain imaging in pigs. METHODS: In vitro receptor autoradiography studies were performed to establish the anatomic distribution of NPY2 receptors in the pig brain. In vivo, baseline 90-min PET recordings of N-(11)C-methyl-JNJ-31020028 were conducted in anesthetized Yorkshire x Landrace pigs, concurrent with arterial blood sampling. Postchallenge scans were conducted after injection of unlabeled JNJ-31020028 as a pharmacologic intervention. Cyclosporine A was used to enhance levels of the PET radiotracer in the brain. The PET images were manually coregistered to a MR imaging atlas of the pig brain. Maps of the N-(11)C-methyl-JNJ-31020028 volume of distribution in the brain were prepared, and regional binding potentials of NPY2 receptors toward the radioligand were calculated using the simplified reference tissue method. RESULTS: In autoradiography studies, N-(11)C-methyl-JNJ-31020028 receptor binding sites were observed primarily in the hippocampus and were inhibited by unlabeled JNJ-31020028. In PET studies, N-(11)C-methyl-JNJ-31020028 was metabolized slowly in the bloodstream, with 25% of the (11)C-labeled parent compound remaining 30 min after injection. PET imaging showed baseline binding potentials of 0.64 ± 0.07 in the thalamus, 0.55 ± 0.02 in the caudate, and 0.49 ± 0.03 in the hippocampus. Graphical reference region analyses demonstrated that N-(11)C-methyl-JNJ-31020028 binding was reversible; infusion of unlabeled JNJ-31020028 markedly displaced the PET radioligand from binding sites in the hippocampus, thalamus, caudate nucleus, and cerebellum but not in the corpus callosum, which served as reference region for nonspecific binding. CONCLUSION: N-(11)C-methyl-JNJ-31020028 has several suitable properties for PET neuroimaging of NPY2 receptors. First, it is metabolized slowly in the bloodstream of pigs. Second, using cyclosporine, the target-to-background ratio of N-(11)C-methyl-JNJ-31020028 is sufficient for estimating pharmacokinetic parameters. Third, N-(11)C-methyl-JNJ-31020028 binds reversibly and competitively to cerebral sites known to contain relatively high numbers of NPY2 receptors, such as the hippocampus, thalamus, caudate nucleus, and cerebellum. Fourth, white matter such as corpus callosum, known to contain negligible numbers of NPY2 receptors, can serve as a reference region for estimating binding potentials in brain regions. To our knowledge, there is no other radioligand with these favorable properties and with this specificity for NPY2 receptors, which makes N-(11)C-methyl-JNJ-31020028 the first candidate radioligand for PET investigations of NPY2 receptors in the living brain.