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.

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.

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.

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.

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.

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.

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.

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.

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.

Top-down mass spectrometry imaging of intact proteins by laser ablation ESI FT-ICR MS.

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation.

MALDI mass spectrometry imaging in microscope mode with infrared lasers: bypassing the diffraction limits.

This letter demonstrates the use of infrared matrix-assisted laser desorption/ionization coupled with microscope mode mass spectrometry imaging. It is aimed to explore the use of intrinsic water in tissue as a matrix for imaging at spatial resolutions below the diffraction limit of the employed IR optics. Stigmatic ion optics with a magnification factor of ~70 were used to project the spatial distribution of produced ions onto a detector while separating ions with different mass-to-charge ratios using a time-of-flight mass spectrometer. A pixelated detector was used to simultaneously record arrival time and impact position. A previously described dried-droplet sample system of 2,5-dihydroxybenzoic acid (DHB) and 5 peptides covered by a copper grid for defined surface structure was used to benchmark the light- and ion-optical setup for spatial resolution and mass spectrometric performance. A spatial resolving power of 9.8 µm, well below the optical limit of diffraction (14 µm for the given setup), was established. After, frozen cryo-sections from a biological model system were measured by exploiting the endogenous water content as a matrix. Principal component analysis enabled a clear distinction between distinct tissue regions identified by both light microscopy and MS imaging.

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.

Absorption mode FTICR mass spectrometry imaging.

Fourier transform ion cyclotron resonance mass spectrometry offers the highest mass resolving power for molecular imaging experiments. This high mass resolving power ensures that closely spaced peaks at the same nominal mass are resolved for proper image generation. Typically higher magnetic fields are used to increase mass resolving power. However, a gain in mass resolving power can also be realized by phase correction of the data for absorption mode display. In addition to mass resolving power, absorption mode offers higher mass accuracy and signal-to-noise ratio over the conventional magnitude mode. Here, we present the first use of absorption mode for Fourier transform ion cyclotron resonance mass spectrometry imaging. The Autophaser algorithm is used to phase correct each spectrum (pixel) in the image, and then, these parameters are used by the Chameleon work-flow based data processing software to generate absorption mode "Datacubes" for image and spectral viewing. Absorption mode reveals new mass and spatial features that are not resolved in magnitude mode and results in improved selected ion image contrast.

Cluster secondary ion mass spectrometry microscope mode mass spectrometry imaging.

RATIONALE: Microscope mode imaging for secondary ion mass spectrometry is a technique with the promise of simultaneous high spatial resolution and high-speed imaging of biomolecules from complex surfaces. Technological developments such as new position-sensitive detectors, in combination with polyatomic primary ion sources, are required to exploit the full potential of microscope mode mass spectrometry imaging, i.e. to efficiently push the limits of ultra-high spatial resolution, sample throughput and sensitivity. METHODS: In this work, a C60 primary source was combined with a commercial mass microscope for microscope mode secondary ion mass spectrometry imaging. The detector setup is a pixelated detector from the Medipix/Timepix family with high-voltage post-acceleration capabilities. The system's mass spectral and imaging performance is tested with various benchmark samples and thin tissue sections. RESULTS: The high secondary ion yield (with respect to 'traditional' monatomic primary ion sources) of the C60 primary ion source and the increased sensitivity of the high voltage detector setup improve microscope mode secondary ion mass spectrometry imaging. The analysis time and the signal-to-noise ratio are improved compared with other microscope mode imaging systems, all at high spatial resolution. CONCLUSIONS: We have demonstrated the unique capabilities of a C60 ion microscope with a Timepix detector for high spatial resolution microscope mode secondary ion mass spectrometry imaging.

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging.

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C60 primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 µm spatial resolution (75 µm primary ion spot size) with mass resolving power (m/Δm(50%)) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm(50%) > 3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 µm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

Lessons of history in veterinary medicine.

The future of veterinary medicine is best understood in the context of history. What began as a profession rooted in urban centers in proximity to horses, physicians, and medical schools, was transformed into a land grant-based agricultural profession with the arrival of the internal combustion engine in the early twentieth century. Most of the United States' current veterinary colleges are still located in towns or small cities in the middle section of the country, outside the largest metropolitan areas where most veterinarians practice companion-animal medicine. Throughout veterinarian history, substantial numbers of US students have been educated in foreign colleges and this continues today, creating an even greater geographic imbalance between the veterinary educational process and US population centers and major medical schools. Three themes deserve special attention as we celebrate the profession's 150th anniversary. We must first move beyond the land-grant culture and develop a more geographically balanced approach to establishing new veterinary colleges that are also in closer association with schools of medicine and public health. We must also facilitate more opportunities for women leadership in organized veterinary medicine, in practice ownership, in academia, and in the corporate structures that educate, hire, and interface with veterinarians. Finally, we need to expand our understanding of One Health to include the concept of zooeyia (the role of animals in promoting human health), as well as continue to emphasize veterinarians' special roles in the control and management of zoonotic diseases and in advancing comparative medicine in the age of the genome.

Enhanced detection of high-mass proteins by using an active pixel detector.

Flying high: Application of an active pixel detector with high charge sensitivity to a linear time-of-flight mass spectrometer results in enhanced detection of high-mass proteins (such as Immunoglobulin G; IgG) using a conventional microchannel plate detection system. This technique thus provides a means to extend the mass range of such detectors as well as allowing direct visualization of mass-dependent ion-focusing phenomena.

C60 secondary ion Fourier transform ion cyclotron resonance mass spectrometry.

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100 000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 µm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.