LibGen: Generating High Quality Spectral Libraries of Natural Products for EAD-, UVPD-, and HCD-High Resolution Mass Spectrometers.

Compound annotation using spectral-matching algorithms is vital for (MS/MS)-based metabolomics research, but is hindered by the lack of high-quality reference MS/MS library spectra. Finding and removing errors from libraries, including noise ions, is mostly done manually. This process is both error-prone and time-consuming. To address these challenges, we have developed an automated library curation pipeline, LibGen, to universally build novel spectral libraries. This pipeline corrects mass errors, denoises spectra by subformula assignments, and performs quality control of the reference spectra by calculating explained intensity and spectral entropy. We employed LibGen to generate three high-quality libraries with chemical standards of 2241 natural products. To this end, we used an IQ-X orbital ion trap mass spectrometer to generate 1947 classic high-energy collision dissociation spectra (HCD) as well as 1093 ultraviolet-photodissociation (UVPD) mass spectra. The third library was generated by an electron-activated collision dissociation (EAD) 7600 ZenoTOF mass spectrometer yielding 3244 MS/MS spectra. The natural compounds covered 140 chemical classes from prenol lipids to benzypyrans with >97% of the compounds showing <0.2 Tanimoto-similarity, demonstrating a very high structural variance. Mass spectra showed much higher information content for both UVPD- and EAD-mass spectra compared to classic HCD spectra when using spectral entropy calculations. We validated the denoising algorithm by acquiring MS/MS spectra at high concentration and at 13-fold diluted chemical standards. At low concentrations, a higher proportion of spectra showed apparent fragment ions that could not be explained by subformula losses of the parent molecule. When more than 10% of the total intensity of MS/MS fragments was regarded as noise ions, spectra were considered as low quality and were not included in the libraries. As the overall process is fully automated, LibGen can be utilized by all researchers who create or curate mass spectral libraries. The libraries we created here are publicly available at MassBank.us.

Acyl-CoA Identification in Mouse Liver Samples Using the In Silico CoA-Blast Tandem Mass Spectral Library.

Acyl-coenzyme A derivatives (acyl-CoAs) are core molecules in the fatty acid and energy metabolism across all species. However, in vivo, many other carboxylic acids can form xenobiotic acyl-CoA esters, including drugs. More than 2467 acyl-CoAs are known from the published literature. In addition, more than 300 acyl-CoAs are covered in pathway databases, but as of October 2020, only 53 experimental acyl-CoA tandem mass spectra are present in NIST20 and MoNA libraries to enable annotation of the mass spectra in untargeted metabolomics studies. The experimental spectra originated from low-resolution ion trap and triple quadrupole mass spectrometers as well as high-resolution quadrupole-time of flight and orbital ion trap instruments at various collision energies. We used MassFrontier software and the literature to annotate fragment ions to generate fragmentation rules and intensities for the different instruments and collision energies. These rules were then applied to 1562 unique species based on [M+H]+ and [M-H]- precursor ions to generate two mass spectra per instrument platform and collision energy, amassing an in silico library of 10,934 accurate mass MS/MS spectra that are freely available at github.com/urikeshet/CoA-Blast. The spectra can be imported into a commercial or freely available mass spectral search tool. We used the libraries to annotate 23 acyl-CoA esters in mouse liver, including 8 novel species.

Doubly Charged Molecular Ions in GC-MS with Cold EI.

We report the finding of doubly charged molecular ions in a range of relatively large molecules including hydrocarbons upon their electron ionization as vibrationally cold molecules in supersonic molecular beams (SMB) (also named as Cold EI). Furthermore, we also report the detection by mass spectrometry of triply charged molecular ions in large PAHs such as decacyclene and ovalene upon their cooling in SMB. We found that the relative abundance of doubly charged molecular ions strongly depends on the internal vibrational cooling. While after some vibrational cooling the fragmentation pattern became cooling independent, the relative abundance of the doubly charged molecular ions was noticeably increased upon further cooling via increasing of the cooling make-up gas flow rate. In addition, the relative abundance of the doubly charged molecular ions was strongly increased with the compounds' size, and its electron energy threshold was lower than expected. These observations indicate a new mechanism that involves two separate electron ionization processes in the same compound, most likely with the same electron but at two separate atoms (places) in large molecules, to reduce Coulombic repulsion energy that can lead to fragmentation into two singly charged ions. These findings are shedding new light on electron ionization mass spectra. Accordingly, electron ionization mass spectra are the result of three separate mechanisms with relative magnitudes that depend on the compound size: (a) single electron ionization; (b) double electron ionization; and (c) single electron ionization with subsequent internal excitation by the same ionizing electron in another place.

Pesticide analysis by pulsed flow modulation GCxGC-MS with Cold EI-an alternative to GC-MS-MS.

We explored the use of pulsed flow modulation (PFM) two-dimensional comprehensive gas chromatography (GCxGC) mass spectrometry with supersonic molecular beams (SMB) (also named Cold electron ionization (EI)) for achieving universal pesticide analysis in agricultural products. The use of GCxGC serves as an alternative to MS-MS in the needed reduction of matrix interference while enabling full-scan MS operation for universal pesticide analysis with reduced number of false negatives. Matrix interference is further reduced with Cold EI in view of the enhancement of the molecular ions. Pulsed flow modulation is a simple GCxGC modulator that does not consume cryogenic gases while providing tuneable second GCxGC column injection time for enabling the use of quadrupole-based mass spectrometry regardless its limited scanning speed. PFM-GCxGC-MS with Cold EI combines improved separation of GCxGC with Cold EI benefits of tailing-free ultra-fast ion source response time and enhanced molecular ions for the provision of increased sample identification information and reduced matrix interference. Consequently, PFM GCxGC-MS with Cold EI also improved NIST library identification probabilities of the spiked pesticides. PFM GCxGC is further characterized by largely increased second column sample and matrix capacity that as a result performs much better than thermal modulation GCxGC-MS with standard EI in the suppression of matrix interference. In a comparison with standard GC-MS, we measured with PFM GCxGC-MS with Cold EI an average total ion count matrix interference reduction factor of 32 for 12 pesticides in two matrices of baby leaves mixture and lettuce. In addition, Cold EI further increases the range of pesticides amenable for GC-MS analysis and its response is relatively uniform hence with it the need for pesticides specific calibration is reduced. Graphical abstract Pulsed flow modulation GCxGC-MS with Cold EI significantly reduces matrix interference and improves sample identification.

Soft Cold EI - approaching molecular ion only with electron ionization.

RATIONALE: Cold EI is defined as electron ionization of cold molecules in supersonic molecular beams (SMB). Gas chromatography/mass spectrometry (GC/MS) with Cold EI provides informative mass spectra, which combine the usual library-searchable EI fragment ions with enhanced molecular ions for improved library-based identification probabilities. However, in some cases, such as in the analysis of complex petrochemical matrices, a soft ionization method that provides only molecular ions is desirable. METHODS: GC/MS with Cold EI was used with a fly-through ion source at selected electron energies, including at low electron energies, in an attempt to observe molecular ions alone. RESULTS: We explored low electron energy Cold EI and found that once the sample compound is cooled by the supersonic expansion it can be reheated via reflected scattered helium atoms near the skimmer. Furthermore, once a labile molecular ion is formed it can undergo undesirable collision-induced dissociation (CID) in the same way as in tandem mass spectrometry (MS/MS), and the magnitude of such CID can be significant for labile molecular ions such as those of hydrocarbons. In order to reduce these adverse effects we reduced the helium pressure at the ion source and MS vacuum chamber by increasing the nozzle-skimmer distance. Cold EI at low electron energies was explored with a squalane isomer (C30 H62 ) and with n-C24 H50 . CONCLUSIONS: It was found that an increased nozzle-skimmer distance resulted in a noticeable increase in the abundance ratio of molecular ions to low mass fragment ions. Consequently, Cold EI at low electron energies and a large nozzle-skimmer distance converts EI into Soft Cold EI while further approaching the ideal of a molecular ion only ionization method.

Strong magnetization measured in the cool cores of galaxy clusters.

Tangential discontinuities, seen as x-ray edges known as cold fronts (CFs), are ubiquitous in cool-core galaxy clusters. We analyze all 17 deprojected CF thermal profiles found in the literature, including three new CFs we tentatively identify (in clusters A2204 and 2A0335). We discover small but significant thermal pressure drops below all nonmerger CFs, and argue that they arise from strong magnetic fields below and parallel to the discontinuity, carrying 10%-20% of the pressure. Such magnetization can stabilize the CFs, and explain the CF-radio minihalo connection.

Critical conditions for core-collapse supernovae.

The explosion of a core-collapse supernova can be approximated by the breakdown of steady-state solutions for accretion onto a proto-neutron star (PNS). We analytically show that as the neutrino luminosity exceeds a critical value L(c), the neutrinosphere pressure exceeds the hydrostatic limit even for an optimal shock radius R. This yields L(c) [proportionally] M(2)T(2) (with logarithmic corrections) and R [proportionally] M/T, in agreement with numerical results, where M and T are the PNS mass and neutrino temperature, respectively. The near-critical flow can be approximated as a ballistic shell on top of an isothermal layer.

Analytical study of diffusive relativistic shock acceleration.

Particle acceleration in relativistic shocks is studied analytically in the test-particle, small-angle scattering limit, for an arbitrary velocity-angle diffusion function D. The particle spectral index s is found to be sensitive to D, particularly downstream and at certain angles. The analysis, confirmed numerically, justifies and generalizes previous results for isotropic diffusion. It can be used to test collisionless shock models and to observationally constrain D. For example, strongly forward- or backward-enhanced diffusion downstream is ruled out by gamma-ray burst afterglow observations.

Survival probabilities of history-dependent random walks.

We analyze the dynamics of random walks with long-term memory (binary chains with long-range correlations) in the presence of an absorbing boundary. An analytically solvable model is presented, in which a dynamical phase transition occurs when the correlation strength parameter mu reaches a critical value mu(c). For strong positive correlations, mu > mu(c), the survival probability is asymptotically finite, whereas for mu < mu(c) it decays as a power law in time (chain length).

Energy spectrum of particles accelerated in relativistic collisionless shocks.

We analytically study diffusive particle acceleration in relativistic, collisionless shocks. We find a simple relation between the spectral index s and the anisotropy of the momentum distribution along the shock front. Based on this relation, we obtain s=(3beta(u)-2beta(u)beta(2)(d)+beta(3)(d))/(beta(u)-beta(d)) for isotropic diffusion, where beta(u) (beta(d)) is the upstream (downstream) fluid velocity normalized to the speed of light. This result is in agreement with previous numerical determinations of s for all (beta(u),beta(d)), and yields s=38/9 in the ultrarelativistic limit. The spectrum-anisotropy connection is useful for testing numerical studies and constraining anisotropic diffusion results. It suggests that the spectrum is highly sensitive to the form of the diffusion function for particles traveling along the shock front.

Phase transition in random walks with long-range correlations.

Motivated by recent results in the theory of correlated sequences, we analyze the dynamics of random walks with long-term memory (binary chains with long-range correlations). In our model, the probability for a unit bit in a binary string depends on the fraction of unities preceding it. We show that the system undergoes a dynamical phase transition from normal diffusion, in which the variance D(L) scales as the string's length L, into a superdiffusion phase ( D(L) approximately Lalpha,alpha>1), when the correlation strength exceeds a critical value. We demonstrate the generality of our results with respect to alternative models, and discuss their applicability to various data, such as coarse-grained DNA sequences, written texts, and financial data.