Characterization of Asian lacquers by atmospheric solids analysis probe high resolution tandem mass spectrometry coupled with cyclic ion mobility separation

Coactivator-associated arginine methyltransferase 1 (CARM1), originally defined as a coactivator for steroid receptors, is a member of the protein arginine methyltransferases. Here, we report the discovery and characterization of an automethylation event by AgCARM1, a CARM1 homologue in the mosquito Anopheles gambiae, using top-down high resolution tandem mass spectrometry, which allows fine mapping of modifications in the intact protein accurately and quantitatively without priori knowledge. Unexpectedly, we found that AgCARM1 has already been predominantly dimethylated during its expression in Escherichia coli. A single arginine methylation site, R485, was identified which is conserved among CARM1 in insects. No methylation was observed in the intact AgCARM1(R485K) mutant where R485 is mutated to lysine, which confirms that R485 is the only detectable methylation site. Using AgCARM1 methyltransferase defective mutants, we confirmed that this is an automethylation event and show the automethylation of AgCARM1 occurs intermolecularly. This study represents the first comprehensive characterization of an automethylation event by top-down mass spectrometry. The unexpected high percentage of automethylated recombinant AgCARM1 expressed in E. coli may shed light on other bacterially expressed post-translational modifying enzymes, which could be modified but overlooked in biochemical and structural studies. Top-down high resolution tandem mass spectrometry thus provides unique opportunities for revealing unexpected protein modification, localizing specific modification to one amino acid, and delineating molecular mechanism of an enzyme.

Multiple phosphorylable sites in the Zaire Ebolavirus nucleoprotein evidenced by high resolution tandem mass spectrometry

Pooling strategy to construct in-house high-resolution electrospray ionization tandem mass spectrometry database of drugs

IntroductionLicochalcone A is a chalcone natural product that has been isolated from roots of the licorice species, Glycyrrhiza inflata. It shows antimalarial, anticancer, antibacterial, and antiviral (specifically against influenza neuraminidase) properties in vitro. Since little information is available concerning the human bioavailability and metabolism of licochalcone A, we carried out preclinical in vitro hepatic metabolism studies. Several Phase I and phase II metabolites were observed and characterized using high resolution LC‐MS/MS with accurate mass measurement. Based on fragmentation pathways during high resolution tandem mass spectrometry and LC‐UV analysis, the structures of licochalcone A metabolites were characterized and in some cases identified.MethodsFor phase I metabolism studies, licochalcone A was incubated with human liver microsomes or recombinant cytochrome P450 enzymes and NADPH in phosphate buffer. After reaction, samples were analyzed using high resolution LC‐MS/MS with accurate mass measurement on a Waters Synapt G1 QToF mass spectrometer. High resolution MS was used to determine the elemental compositions of the phase I metabolites, and product ion tandem mass spectrometry was used for structural elucidation.Incubations were also carried out using cryopreserved human hepatocytes to investigate not only phase I metabolism but also phase II conjugation with glucuronic acid, sulfate and glutathione. Deconjugation by using beta‐glucuronidase and sulfatase followed by HPLC‐UV and high resolution tandem mass spectrometry confirmed the formation of phase II conjugates.Preliminary DataAfter incubation with human liver microsomes, five (5) mono‐oxygenated or di‐oxygenated licochalcone A phase I metabolites were detected. When microsomes or NADPH was omitted from the incubation, no metabolites were detected, indicating that oxidative metabolism occurred via cytochrome P450 enzymes. The biotransformation pathway of each phase I metabolite was determined using recombinant cytochrome P450 enzymes. Based on accurate mass MS/MS data and application of Biemann's shift method of data interpretation, addition of oxygen to licochalcone A could be localized on either the A‐ring or on the B‐ring.Incubation of licochalcone A produced several glucuronide metabolites, including glucuronic acid conjugates of phase I oxygenated metabolites. One sulfate conjugate and one GSH conjugate of licochalcone A were also found. Using a combination of MS/MS and UV analysis, the two most abundant mono‐glucuronide conjugates of licochalcone A were identified as 4‐O‐glucuronide and 4′‐O‐glucuronide. The in vitro metabolic stability of licochalcone A was investigated, and the half‐life was determined to be 83 min.ConclusionLicochalcone A, a chemoprevention agent in the licorice species G. inflate, is slowly metabolized by human hepatic cytochrome P450 enzymes to form several mono‐ and di‐oxygenated phase I metabolites. However, phase II glucuronidation followed by sulfation is expected to occur much more rapidly. These in vitro metabolism studies of licochalcone A will be useful in predicting its bioavailability and potential efficacy as a chemoprevention agent in vivo.Support or Funding InformationNIH grant P50 AT000155 from the Office of Dietary Supplements and the National Center for Complementary and Integrative Health. NIH grant R01 AT007659 from the National Center for Complementary and Integrative Health and the Office of the Director.

Simultaneous quantification of neuroactive dopamine serotonin and kynurenine pathway metabolites in gender-specific youth urine by ultra performance liquid chromatography tandem high resolution mass spectrometry

Based on high-resolution mass spectrometry (HRMS), nontarget analysis (NTA) can rapidly identify and characterize numerous hazardous substances in complex environmental samples. However, the intricate identification process often results in the underutilization of many mass spectrometry features. Even when chemical structures are identified, their toxicological effects and health outcomes may remain unknown. To address these challenges, this study introduces MSFragTox, a novel approach that leverages the rich fragmentation spectra inherent in high resolution tandem mass spectrometry (MS/MS) to directly predict toxicity. This method integrates MS/MS data with high-throughput screening (HTS) assays, focusing on seven endocrine disruption-related endpoints from Tox21, and uses MS-derived fingerprints: substructure fragmentation probability vectors to construct toxicity predictions using machine learning algorithms. The best model demonstrated robust performance with an average area under the receiver operating characteristic curve (AUROC) of 0.845 on the test set, outperforming models based on traditional molecular fingerprints and descriptors. Additionally, a web client (http://ms.envwind.site:8500) is provided for users to screen toxicity based on chemical MS/MS data. Furthermore, in-depth analyses of commonalities and differences in substructures reveal the mechanisms underlying across toxicity endpoints. Using MSFragTox, we validated the potential endocrine-disrupting effects of substances corresponding to MS/MS from real samples, highlighting the feasibility of directly studying toxicity through MS/MS and its potential applications in risk prediction and early warning for environmental samples.

Optimized ultra performance liquid chromatography tandem high resolution mass spectrometry method for the quantification of paraquat in plasma and urine.

Cobalt was included on the World Anti-Doping Agency Prohibited List in 2015 due to its effect on stimulus of erythropoiesis via stabilization of hypoxia-inducible factor. Although it has proven benefits for performance enhancement, the unavailability of inductively coupled plasma-mass spectrometry on routine of the accredited laboratories is a factor that reduces its applicability in anti-doping analysis. Therefore, an analytical method for quantification of urinary cobalt as its diethyldithiocarbamate complex by liquid chromatography coupled with high-resolution tandem mass spectrometry was developed and validated. Palladium was proposed as internal standard and rhodium as a complexation control. A microwave-assisted acid digestion of the urine samples was essential, not only to eliminate the matrix effect but mainly to avoid the non-specific bond of cobalt to endogenous molecules. A linear method was obtained over the studied range from a negative urine control to a spiked concentration of 25 ng/mL, with an estimated limit of quantification of 2.5ng/mL, and an adequate combined standard uncertainty of 11.4%. Considering that all reagents are commercially available, the proposed strategy is feasible to be included on routine sample preparation. Monitoring urinary cobalt concentrations globally opens the perspective to support the anti-doping system to define a suitable threshold value and to understand its potential misuse by athletes seeking for performance improvement.

Natamycin is a naturally occurring antimycotic used to prevent mold in a variety of foodstuffs, mainly processed meat, cheese and wine, and more recently certain fresh fruits. The aim of the present study was to develop and validate a sensitive and selective method for the quantification of natamycin residues in blueberries. A methanolic extraction followed by solid phase extraction clean up and analysis by liquid chromatography coupled to high resolution tandem mass spectrometry validated for the analysis of natamycin residues in blueberries following the SANTE guidelines. The method was validated at 0.025, 0.25 and 11 mg/kg with average recoveries between 86 and 105%, and precision (%RSD) was below 15%. The detection limit for natamycin was below 0.010 mg/kg. Residues of natamycin in treated blueberries were found to range from 1 to 29 mg/kg and remained stable over 4 weeks of storage at 4 °C. The LC-HRMS analysis reported here is shown to be an accurate and sensitive method for quantifying natamycin residues in blueberries. A method suitable for use in the regulatory and commercial monitoring of natamycin residues in blueberries is developed and presented.

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.

The mission of the UIC/NIH Center for Botanical Dietary Supplements Research is to evaluate the safety and efficacy of botanical dietary supplements with a focus on women's health. Botanicals are selected for study based on ethnobotanical use and then botanically authenticated, extracted and fractionated for experiments designed to determine mechanisms of action, identification of active constituents, and safety. In support of all of these studies, ultrahigh performance liquid chromatography (UHPLC) in combination with triple quadrupole tandem mass spectrometry or high resolution tandem mass spectrometry is essential for the characterization, identification and quantitative analysis of active constituents and their metabolites. Compared with the iterative process of bioassay-guided fractionation, ultrafiltration high resolution mass spectrometry significantly shortens the time required for the identification of estrogenic or chemopreventive active constituents. Quantitative analysis of active constituents in botanical preparations or human serum in support of pharmacokinetics studies demands the selectivity of mass spectrometry, and the use of UHPLC in place of HPLC enhances the throughput of these assay by up to 12-fold. Finally, UHPLC-MS-MS enhances the productivity of studies of metabolism, metabolic activation, and drug-botanical interactions. Supported by grant P50 AT000155 from the NIH ODS and NCCAM

This study aims to identify and characterize compounds refractory to hydrodenitrogenation (HDN). The efficiency of a vacuum hydrocracking unit in removing nitrogen-containing compounds to produce a low-nitrogen-content effluent is examined. Molecular, structural, and compositional knowledge is a requisite for the optimization development of the hydrotreatment step processing unit because changes in the chemical composition of petroleum have a direct impact on physical properties and, thus, overall vacuum gas oil (VGO) upgrading processes. Two samples, a VGO and a effluent obtained after the HDN process containing 10 wppm of N, were analyzed by negative-mode electrospray ionization ultra-high-resolution tandem mass spectrometry (ESI–FT-ICR) and ion mobility spectrometry–mass spectrometry (IMS–MS). FT-ICR mass spectrometry provides ultrahigh mass resolving power to separate and characterize compounds in the highly complex petroleum samples. The fragments generated by MS/MS of selected N₁ refractory compounds were used for structural elucidation. Species with a double bond equivalent of 10 and 13, which proved to be highly refractory, were analyzed in more detail. On the other hand, the TWIMS–TOF MS measurements enabled the entire ion mobility analysis of refractory species to hydrotreatment processes. The MS/MS spectra revealed a specific pattern allowing for the identification of the molecular nucleus. Furthermore, it allowed for the understanding of the fragmentation pathways: loss of alkyl chains in the first step and opening and rearrangement of the nuclei after that. Ion mobility separation, in combination with MS/MS, allowed for the identification of the different conformations and the revealment of the typical fragments of these molecular nuclei. In particular, the ion mobility peak width indicates isomeric diversity and collision cross section (CCS) determination and provides structural information. The IMS analysis of the identified refractory precursor shows the evolution of its compounds at different processing stages and indicates that some families and structural conformations of N₁ species are more resistant to hydrotreatment. Isomers presenting low CCS values in negative mode are more resistant to HDN processes. The combination of TWIMS–TOF MS and ultra-high-resolution mass spectrometry opens exciting and promising prospects for structural determination of complex mixtures, in particular, problematic compounds in petroleum refining processes.

Advances in high resolution tandem mass spectrometry and peptide enrichment technologies have transformed the field of protein biochemistry by enabling analysis of end points that have traditionally been inaccessible to molecular and biochemical techniques. One field benefitting from this research has been the study of ubiquitin, a 76-amino acid protein that functions as a covalent modifier of other proteins. Seminal work performed decades ago revealed that trypsin digestion of a branched protein structure known as A24 yielded an enigmatic diglycine signature bound to a lysine residue in histone 2A. With the onset of mass spectrometry proteomics, identification of K-GG-modified peptides has emerged as an effective way to map the position of ubiquitin modifications on a protein of interest and to quantify the extent of substrate ubiquitination. The initial identification of K-GG peptides by mass spectrometry initiated a flurry of work aimed at enriching these post-translationally modified peptides for identification and quantification en masse. Recently, immunoaffinity reagents have been reported that are capable of capturing K-GG peptides from ubiquitin and its thousands of cellular substrates. Here we focus on the history of K-GG peptides, their identification by mass spectrometry, and the utility of immunoaffinity reagents for studying the mechanisms of cellular regulation by ubiquitin.

Imaged capillary isoelectric focusing (icIEF) tandem high resolution mass spectrometry for charged heterogeneity of protein drugs in biopharmaceutical discovery

A high resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is used for characterizing the fragmentation of chlorophyll-a. Three tandem mass spectrometry (MS/MS) techniques, including electron-induced dissociation (EID), collisionally activated dissociation (CAD), and infrared mutiphoton dissociation (IRMPD) are applied to the singly protonated chlorophyll-a. Some previously unpublished fragments are identified unambiguously by utilizing high resolution and accurate mass value provided by the FTICR mass spectrometer. According to this research, the two long aliphatic side chains are shown to be the most labile parts, and favorable cleavage sites are proposed. Even though similar fragmentation patterns are generated by all three methods, there are much more abundant peaks in EID and IRMPD spectra. The similarities and differences are discussed in detail. Comparatively, cleavage leading to odd electron species and H(•) loss both seem more common in EID experiments. Extensive loss of small side groups (e.g., methyl and ethyl) next to the macrocyclic ring was observed. Coupling the high performance FTICR mass spectrometer with contemporary MS/MS techniques, especially IRMPD and EID, proved to be very promising for the structural characterization of chlorophyll, which is also suitable for the rapid and accurate structural investigation of other singly charged porphyrinic compounds.