Calculated electron impact ionization fragmentation patterns

There are many measurements and calculations of total electron impact ionisation cross sections. However, many applications, particularly in plasma physics, also require fragmentation patterns. Approximate methods of deducing partial cross sections are tested based on the use of total cross section computed within the well-used Binary Encounter Bethe (BEB) approximation. Partial ionisation cross sections for three series of molecules including CH$_4$, CF$_4$ and CCl$_4$; SiH$_4$ and SiCl$_4$; NH$_3$ and PH$_3$, were estimated using two methods. Method one is semi-empirical and uses mass spectroscopy data to fix the partial cross sections at a single electron energy. The second is a a fully computational method proposed by Huber {\it et al.} (2019, J. Chem. Phys., 150, 024306). Comparisons with experimental results suggest that the mass spectroscopy method is more accurate. However, as Huber's method requires no experimental input, this method could be used as a first approximation when no experimental data is available. As mass spectroscopy sometimes provides incomplete datasets, a hybrid method based on the use of both methods is also explored.

Application of a HPLC-Q/TOF method with post-column compensation for separation and identification of polar impurities in Cytidine disodium triphosphate for injection

Background: Cytidine Disodium Triphosphate (CTP-2Na) for injection is mainly used for treating nervous system diseases. Currently, there are few studies focused on the separation and identification of polar impurities in CTP-2Na for injection, which is important for ensuring drug safety and efficacy. Objective: The study aimed to establish an HPLC-Q/TOF method for the separation and identification of polar impurities in CTP-2Na for injection. Methods: Chromatographic separation was achieved on a Waters Atlantis T3 column using 5 mM aqueous ammonium acetate solution as the mobile phase in an isocratic elution mode. A postcolumn compensation technology was used to improve the ionization efficiency of impurities in the spray chamber. Results: Three polar impurities (disodium cytidine tetraphosphate, disodium cytidine diphosphate, disodium cytidine monophosphate) were detected in CTP-2Na for injection. The former one is probably the overreaction product during the production of CTP-2Na, the latter two were reported as degradation products. The fragmentation patterns of cytidine phosphate compounds in negative ion mode are summarized. Conclusion: This study provides a good reference for the separation and identification of polar impurities in nucleotide drugs.

Identification of a Novel Secreted Metabolite Cyclo(Phenylalanyl-Prolyl) from Batrachochytrium Dendrobatidis and its effect on Galleria Mellonella.

The fungus, Batrachochytrium dendrobatidis, is the causative agent of chytridiomycosis and a leading cause of global decline in amphibian populations . The first stages of chytridiomycosis include: inflammation, hyperkeratosis, lethargy, loss of righting reflex, and disruption of internal electrolyte levels leading to eventual death of the host. Previous work indicates that B. dendrobatidis can produce immunomodulatory compounds and other secreted molecules that regulate the growth of the fungus. In this study, filtrates of the fungus grown in media and water were subjected to ultra performance liquid chromatography-mass spectrometry and analyzed using Compound Discoverer 3.0. Identification of cyclo(phenylalanyl-prolyl), chitobiose, and S-adenosylmethionine were verified by their retention times and fragmentation patterns from B. dendrobatidis supernatants. Previous studies have analyzed the effects of B. dendrobatidis on amphibian models, in vitro, or in cell culture. We studied the effects of live B. dendrobatidis cells, spent culture filtrates containing secreted metabolites, and cyclo(pheylalanyl-prolyl) on wax moth larvae ( Galleria mellonella) . Concentrated filtrates caused melanization within 24 hours, while live B. dendrobatidis caused melanization within 48 hours. Our results indicate B. dendrobatidis produces secreted metabolites previously unreported. These findings provide another alternative for the use of a non-amphibian model system to study pathogenicity traits in this fungus.

At the dawn: cell-free DNA fragmentomics and gene regulation

Epigenetic mechanisms play instrumental roles in gene regulation during embryonic development and disease progression. However, it is challenging to non-invasively monitor the dynamics of epigenomes and related gene regulation at inaccessible human tissues, such as tumours, fetuses and transplanted organs. Circulating cell-free DNA (cfDNA) in peripheral blood provides a promising opportunity to non-invasively monitor the genomes from these inaccessible tissues. The fragmentation patterns of plasma cfDNA are unevenly distributed in the genome and reflect the in vivo gene-regulation status across multiple molecular layers, such as nucleosome positioning and gene expression. In this review, we revisited the computational and experimental approaches that have been recently developed to measure the cfDNA fragmentomics across different resolutions comprehensively. Moreover, cfDNA in peripheral blood is released following cell death, after apoptosis or necrosis, mainly from haematopoietic cells in healthy people and diseased tissues in patients. Several cfDNA-fragmentomics approaches showed the potential to identify the tissues-of-origin in cfDNA from cancer patients and healthy individuals. Overall, these studies paved the road for cfDNA fragmentomics to non-invasively monitor the in vivo gene-regulatory dynamics in both peripheral immune cells and diseased tissues.

A flexible workflow for building spectral libraries from narrow window data independent acquisition mass spectrometry data

Advances in library-based methods for peptide detection from data independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for accurate peptide detection and quantitation in wide window DIA data. We compare the results of this search workflow to other library-free approaches and demonstrate that our search is competitive in terms of accuracy and sensitivity. These results demonstrate that the proposed workflow has the capacity to generate spectral libraries while avoiding the limitations of other methods.

2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards

Compounds from the N-benzylphenethylamine (NBPEA) class of novel psychoactive substances are being increasingly utilized in neurobiological and clinical research, as diagnostic tools, or for recreational purposes. To understand the pharmacology, safety, or potential toxicity of these substances, elucidating their metabolic fate is therefore of the utmost interest. Several studies on NBPEA metabolism have emerged, but scarce information about substances with a tetrahydrobenzodifuran (“Fly”) moiety is available. Here, we investigated the metabolism of 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b’]difuran-4-yl)-N-(2-methoxybenzyl)ethan-1-amine (2C-B-Fly-NBOMe) in three different systems: isolated human liver microsomes, Cunninghamella elegans mycelium, and in rats in vivo. Phase I and II metabolites of 2C-B-Fly-NBOMe were first detected in an untargeted screening and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Several hypothesized metabolites were then synthesized as reference standards; knowledge of their fragmentation patterns was utilized for confirmation or tentative identification of isomers. Altogether, thirty-five phase I and nine phase II 2C-B-Fly-NBOMe metabolites were detected. Major detected metabolic pathways were mono- and poly-hydroxylation, O-demethylation, oxidative debromination, and to a lesser extent also N-demethoxybenzylation, followed by glucuronidation and/or N-acetylation. Differences were observed for the three used media. The highest number of metabolites and at highest concentration were found in human liver microsomes. In vivo metabolites detected from rat urine included two poly-hydroxylated metabolites found only in this media. Mycelium matrix contained several dehydrogenated, N-oxygenated, and dibrominated metabolites.

Noninvasive Cell-of-Origin Classification of Diffuse Large B-Cell Lymphoma Using Inferred Gene Expression from Cell-Free DNA Sequencing

Background Diffuse large B-cell lymphoma (DLBCL) is a genetically and clinically heterogeneous disease. The cell-of-origin (COO) classification subdivides DLBCL into the transcriptionally defined activated B-cell (ABC) and germinal center B-cell (GCB) subtypes. While RNA based methods are considered the gold standard to determine COO, they are rarely used in clinical routine due to logistical and methodological challenges. Alternatives include the immunohistochemistry-based Hans algorithm and classifiers to infer the COO subtype from DNA sequencing [Esfahani et al., Blood 2019]. Despite the undisputed value of these methods, the concordance with the gold standard RNA and their prognostic implication are limited. We have recently shown that expression of individual genes can be inferred from cfDNA fragmentation patterns using a method called EPIC-Seq (EPigenetic expression Inference from Cell-free DNA Sequencing) [Esfahani et al., Cancer Res. 2020]. We therefore reasoned that EPIC-Seq may improve COO classification compared to other non-RNA methods. Methods A gene expression model, using cfDNA fragmentation patterns, was trained using leukocyte RNA-sequencing and deep whole genome profiling of the plasma cell-free DNA of an individual with no evidence of circulating disease. The trained model takes two features into account to infer gene expression: 1. promoter fragmentation entropy (PFE), and 2. normalized coverage at the nucleosome-depleted region of a given transcription start site (TSS). We then used a capture panel targeting TSS specifically designed for EPIC-Seq. We selected genes based on their power to discriminate COO subtypes in tumor RNA sequencing [Schmitz et al., NEJM 2018]. We first developed a classifier to distinguish DLBCL cases from healthy plasma using the inferred gene expression. Moreover, we defined GCB and ABC signature scores as the average inferred expression of a set of 'GCB genes' (n=34) and 'ABC genes' (n=34), respectively. Finally, we defined the COO score as the difference between the GCB and ABC scores. To validate our assay and method, we profiled 71 plasma samples from 68 healthy individuals and 90 pretreatment plasma samples from patients with large B-cell lymphomas using EPIC-Seq. Results We first evaluated the performance of the EPIC-Seq classifier in distinguishing DLBCL cases from controls and achieved an AUC of 0.92 in a cross-validation setting (Fig 1a). We then compared the result of EPIC-Seq COO classifier with the genotype-based method previously developed in our group. We observed epigenetic scores to be significantly correlated with previously described mutation-based GCB scores (r=0.75, P=1E-5, Fig. 1b). When comparing to the Hans classification algorithm, we observed significantly higher GCB scores in cases classified as GCB by Hans as compared with non-GCB cases (Wilcox P=0.001, Fig. 1c). Comparing the prognostic power of epigenetic and mutation-based COO labels in previously untreated patients using univariate Cox regressions, the EPIC-Seq classifier better stratified event-free survival (EFS) with higher GCB scores being associated with favorable outcomes (n=70, EPIC-Seq: HR=0.13, P=0.033 vs CAPP-Seq: HR=0.95, P=0.62). Importantly, when binarizing patients into GCB and non-GCB cases by the median, patients with tumors classified as GCB had significantly longer EFS than non-GCB counterparts (log-rank P=0.013, Fig. 1d). The Hans algorithm, in contrast, failed to stratify patients for EFS, among patients analyzed by both immunohistochemistry and DNA genotyping (Fig. 1e). Finally, we profiled n=12 additional DLBCL cases by both RNA-sequencing and EPIC-Seq. Strikingly, we found EPIC-COO scores to be significantly correlated with RNA based GCB scores (r=0.84, P=6E-4, Fig. 1f) underscoring the concordance of EPIC-Seq based COO classification with a gold standard scoring system. Conclusions We here apply EPIC-Seq, a method to infer expression of individual genes from cfDNA fragmentation patterns, to classify DLBCL patients into COO subtypes. COO classification by EPIC-Seq outperformed both Hans and mutation-based methods with regards to outcome stratification and correlated well with RNA-based methods. Overall, these results suggest that EPIC-Seq has utility for noninvasive classification of DLBCL cell-of-origin subtypes and may help to overcome logistical and methodological challenges of RNA-based methods. Figure 1 Figure 1. Disclosures Shahrokh Esfahani: Foresight Diagnostics: Current holder of stock options in a privately-held company. Kurtz: Genentech: Consultancy; Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company; Roche: Consultancy. Diehn: Foresight Diagnostics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CiberMed: Current holder of stock options in a privately-held company, Patents & Royalties; Illumina: Research Funding; Varian Medical Systems: Research Funding; BioNTech: Consultancy; RefleXion: Consultancy; AstraZeneca: Consultancy; Roche: Consultancy. Alizadeh: Gilead: Consultancy; Bristol Myers Squibb: Research Funding; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

UHPLC-ESI-QTOF-MS Analysis on Canthium Coromandelicum Stem

A rapid ultra high-performance liquid chromatography coupled with crossover triple quadrupole time of flight mass spectrometry (UHPLC- ESI -QTOF-MS/MS) method has been developed for the identification of debasement products. According to the distinctive fragmentation patterns, the presence of 79 compounds with retention time between 1.05 to 26.81 minutes was found. In Canthium coromandelicum stem, 22 amino acids, 10 fatty acids, 6 alkaloids, 6 steroids, 2 flavonoids, 2 terpenoids, 2 phenolic, 4 lipids, 3 anthraquinone glycosides, sugars, vitamins were distinguished. These outcomes demonstrated that the contemporary technique has been utilized for quality control of Canthium coromandelicum, exceptionally for recognizable proof, verification and portrayal in medication arrangements.

Metabolite Profiling of Meridianin C In Vivo of Rat by UHPLC/Q-TOF MS

Meridianin C (MC), as a marine alkaloid, is a potent protein kinase inhibitor which exhibits good anticancer activity. However, the in vivo metabolism of MC has not been described to date. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method is employed to investigate the in vivo metabolites of MC in rats. Plasma, bile, urine, and feces are collected after a single oral dose of MC. Protein precipitation, solid phase extraction (SPE), and ultrasonic extraction methods are used to prepare samples. Based on the mass spectral fragmentation patterns, elution order, and retrieving literatures, a total of 13 metabolites of MC were detected and tentatively identified, utilizing MetaboLynx software. The metabolic pathways of MC in rats include N- or O-glucuronidation, O-sulfation, N-hydroxylation, dihydroxylation, and trihydroxylation. The relative content of the metabolites in each kinds of biological samples is also evaluated. This study will help to understand the in vivo properties of MC for the future usage.