The mycelium of the Trametes versicolor synn. Coriolus versicolor (Turkey tail mushroom) exhibit anti-melanoma activity in vitro.

Melanoma is one of the most aggressive forms of skin cancer and is characterized by high metastatic potential. Despite improvements in early diagnosis and treatment, the mortality rate among metastatic melanoma patients continues to represent a significant clinical challenge. Therefore, it is imperative that we search for new forms of treatment. Trametes versicolor is a mushroom commonly used in Chinese traditional medicine due to its numerous beneficial properties. In the present work, we demonstrate T. versicolor fruiting body and mycelium ethanol extracts exhibit potent cytotoxic activity towards A375 (IC50 = 663.3 and 114.5 µg/mL respectively) and SK-MEL-5 (IC50 = 358.4 and 88.6 µg/mL respectively) human melanoma cell lines. Further studies revealed that T. versicolor mycelium extract induced apoptotic cell death and poly (ADP-ribose) polymerase cleavage, upregulated the expression of autophagy-associated marker LC3-II, increased the presentation of major histocompatibility complex II and expression of programmed death-ligand receptor, and inhibited cell migration in SK-MEL-5 cells. Therefore, our present findings highlight the therapeutic potential of T. versicolor mycelium extract for the treatment of melanoma and merit further study.

THE DEVELOPMENT OF A HIGH-RESOLUTION MASS SPECTROMETRY METHOD FOR ULTRA-TRACE ANALYSIS OF CHLORINATED DIOXINS IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES INCLUDING VIET NAM ERA VETERANS.

Chlorinated dioxins are labeled and recognized by both the World Health Organization and the United Nations Environmental Programme (UNEP) as "persistent organic pollutants". Their potential for high toxicity is one of the primary factors behind intense public and regulatory scrutiny and the need to measure the compounds at very low limits, specifically the isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). This article highlights the early mass spectrometry methods to investigate, detect, confirm, and quantify chlorinated dioxins and the initial applications involving human biomonitoring, as attempts were made to attribute health effects to TCDD exposure. This effort represented a complex and difficult scientific response to the pressing need to investigate expected exposures and alleged subsequent medical effects, which in the case of the Viet Nam veterans was being attempted a decade or more after their exposure. It is noteworthy that this method and its development touched on delicate issues involving human subjects, war veterans, environmental contamination, and was difficult not only scientifically, but for ethical and political reasons as well. Stable-isotope dilution with analysis by gas chromatography/high-resolution mass spectrometry (GC/HRMS) became the method of choice because of its ability to monitor characteristic ions and isotope ratios to quantify and qualify/confirm the analyte in the presence of coextracting and coeluting interferences at these low levels with the highest possible confidence. This method was rigorously tested and validated before it was used to discover and monitor levels in the environment and in various populations at then unprecedented low levels. These early studies demonstrated the feasibility of monitoring dioxins in humans even decades after exposure, and led to the detection of 2,3,7,8-TCDD in the general population as well as specific overexposed populations. These studies also provided strong evidence regarding the origins of the 2,3,7,8-isomer in the environment. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Matrix-assisted ionization Fourier transform mass spectrometry for the analysis of lipids.

RATIONALE: Assessing the utility of vacuum matrix-assisted ionization (MAI) for the direct and rapid analysis of lipids in complex samples with emphasis on bacterial taxonomy. METHODS: Matrix-assisted ionization Fourier transform mass spectrometry (MAI-FTMS) was used to characterize polar and non-polar lipids in mixtures. RESULTS: For non-polar lipid triacylglycerols (TAGs), MAI-FTMS produced lipid-specific ions for eight different edible oils and allowed these oils to be identified based on their MAI-FTMS profiles. For polar lipids from bacteria, MAI-FTMS of crude lipid extracts allowed taxonomic identification of eight blind-coded samples based on taxonomy-specific phospholipid profiles. MAI produced results comparable and complementary to benchmark MALDI and ESI methods currently used for characterization of polar and non-polar lipids in the same mixtures. CONCLUSIONS: The newly developed MAI technique is a rapid, simple and complementary method for the characterization of polar and non-polar lipids in complex mixtures.

Gas chromatography-mass spectrometry of JWH-018 metabolites in urine samples with direct comparison to analytical standards.

JWH-018 (1-pentyl-3-(1-naphthoyl)indole) is one of numerous potential aminoalkylindoles contained in products marketed as 'K2' or 'Spice'. Investigation of the urinary metabolites from consumption of these compounds is important because they are banned in the United States and many European countries. An efficient extraction procedure and gas chromatography-mass spectrometry (GC-MS) method were developed for detection of 'K2' metabolites in urine from individuals suspected of using these products. Analytical standards were used to elucidate the structure-specific mass spectral fragmentations and retention properties to confirm proposed identifications and support quantitative studies. A procedure for the synthesis of one of these metabolites (5-hydroxypentyl JWH-018) was also developed. Results are comparable to existing LC-MS/MS methods, with the same primary metabolites detected. The specific metabolite hydrolysis products include 4-hydroxpentyl, 5-hydroxypentyl, and N-pentanoic acid derivatives.

Separation of xylose oligomers using centrifugal partition chromatography with a butanol-methanol-water system.

Xylose oligomers are the intermediate products of xylan depolymerization into xylose monomers. An understanding of xylan depolymerization kinetics is important to improve the conversion of xylan into monomeric xylose and to minimize the formation of inhibitory products, thereby reducing ethanol production costs. The study of xylan depolymerization requires copious amount of xylose oligomers, which are expensive if acquired commercially. Our approach consisted of producing in-house oligomer material. To this end, birchwood xylan was used as the starting material and hydrolyzed in hot water at 200 °C for 60 min with a 4 % solids loading. The mixture of xylose oligomers was subsequently fractionated by a centrifugal partition chromatography (CPC) with a solvent system of butanol:methanol:water in a 5:1:4 volumetric ratio. Operating in an ascending mode, the butanol-rich upper phase (the mobile phase) eluted xylose oligomers from the water-rich stationary phase at a 4.89 mL/min flow rate for a total fractionation time of 300 min. The elution of xylose oligomers occurred between 110 and 280 min. The yields and purities of xylobiose (DP 2), xylotriose (DP 3), xylotetraose (DP 4), and xylopentaose (DP 5) were 21, 10, 14, and 15 mg/g xylan and 95, 90, 89, and 68 %, respectively. The purities of xylose oligomers from this solvent system were higher than those reported previously using tetrahydrofuran:dimethyl sulfoxide:water in a 6:1:3 volumetric ratio. Moreover, the butanol-based solvent system improved overall procedures by facilitating the evaporation of the solvents from the CPC fractions, rendering the purification process more efficient.

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured and denatured protein molecules are equilibrated at easily detectable ratios, namely 1:1. It also requires that closed and open states have lifetimes measurable in the time frame of the HDX experiment. Because both conditions are met by PEPS, these measurements can provide an accurate assessment of closed/open state populations and thus protein folding energies/rates.

Laser desorption/ionization time-of-flight mass spectrometry of triacylglycerols and other components in fingermark samples.

The chemical composition of fingermarks could potentially be important for determining investigative leads, placing individuals at the time of a crime, and has applications as biomarkers of disease. Fingermark samples containing triacylglycerols (TAGs) and other components were analyzed using laser desorption/ionization (LDI) time-of-flight mass spectrometry (TOF MS). Only LDI appeared to be useful for this application while conventional matrix-assisted LDI-TOF MS was not. Tandem MS was used to identify/confirm selected TAGs. A limited gender comparison, based on a simple t-distribution and peaks intensities, indicated that two TAGs showed gender specificity at the 95% confidence level and two others at 97.5% confidence. Because gender-related TAGs differences were most often close to the standard deviation of the measurements, the majority of the TAGs showed no gender specificity. Thus, LDI-TOF MS is not a reliable indicator of gender based on fingermark analysis. Cosmetic ingredients present in some samples were identified.

Separation and purification of xylose oligomers using centrifugal partition chromatography.

Xylose oligomers, which have a prebiotic effect, have been used as additives to human and animal food. These oligomers are also the primary intermediate in hemicellulose degradation during the pretreatment of biomass. Centrifugal partition chromatography (CPC) was used in this study to separate and purify xylan-derived oligomers from birchwood xylan. The xylan was partially hydrolyzed to achieve varying degrees of polymerization at 130°C using 0.98% aqueous sulfuric acid for 20 min with a 2.5% solid loading. The CPC solvent system consisting of dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and water in a 1:6:3 volumetric ratio was used because of its ability to dissolve xylose oligomers of different degrees of polymerization. The CPC was operated in the ascending mode with the water- and DMSO-rich bottom phase acting as the stationary phase, while the THF-rich top phase was the eluent. This paper delineates a method for the production and purification of xylose monomer and xylose oligomers (up to xylopentaose) using CPC. The amount and purity of compounds collected from the CPC fractionation based on 1 g of birchwood xylan were 25.26 mg of xylose at 91.86% purity, 10.71 mg of xylobiose at 85.07% purity, 4.15 mg of xylotriose at 54.71% purity, 5.03 mg of xylotetraose at 38.33% purity and 3.31 mg of xylopentaose at 30.43% purity.

A rapid separation technique for overcoming suppression of triacylglycerols by phosphatidylcholine using MALDI-TOF MS.

Phospholipids and triacylglycerols (TAGs) are important classes of lipids in biological systems. Rapid methods have been developed for their characterization in crude samples, including MALDI time-of-flight MS. For mixtures, MALDI often selectively shows only some components. For example, phosphatidylcholine (PC) suppresses detection of other lipids. Most rapid MS methods detect either TAGs or phospholipids but not both. Herein, we demonstrate a simple approach to rapidly screen mixtures containing multiple lipid classes. To validate this approach, reference lipids [PC, tripalmitin (PPP), and phosphatidyl-ethanolamine (PE)] and real samples (beef, egg yolk) were used. In a binary mixture with a strong suppressor (PC), PPP was greatly suppressed. After a simple separation, suppression was virtually eliminated. A mixture of nominally nonsuppressing lipids (PE and PPP) was not adversely affected by separation. Ground beef and egg yolk were used to demonstrate detection of known lipid compositions where other methods have missed one or more lipids or lipid classes. Separation was performed using solid phase extraction with a PrepSep florisil column. A 10 min separation allows rapid screening for lipids and changes in lipids. It is sufficient to clearly detect all lipids and overcome suppression effects in complex lipid mixtures.

Switchgrass water extracts: extraction, separation and biological activity of rutin and quercitrin.

Switchgrass (Panicum virgatum L.) has recently received significant attention as a possible feedstock for the production of liquid fuels such as ethanol. In addition, switchgrass may also be a source of valuable co-products, such as antioxidants, and our laboratory recently reported that switchgrass contains policosanols and alpha-tocopherol. Motivation for this work began when a switchgrass sample was extracted with water at 50 degrees C and was then tested for low-density lipoprotein (LDL) oxidation inhibition activity using the Thiobarbituric Acid Reactive Substances (TBARS) assay. The TBARS results showed that the switchgrass water extracts inhibited LDL oxidation by as much as 70% in comparison to the control. Liquid chromatography coupled with mass spectrometry (LC-MS) and high performance liquid chromatography (HPLC) were used to identify the compounds that were responsible for LDL oxidation inhibition activity as flavonoids: quercitrin (quercetin-3-O-rhamnoside) and rutin (quercetin-3-O-rutinoside). To maximize flavonoid concentrations, switchgrass was then extracted with water and 60% methanol at different temperatures. The 60% methanol treatment resulted in higher rutin and quercitrin yields when compared to water-only extraction; however, the use of this solvent would not be practical with current biorefinery technology. Centrifugal partition chromatography (CPC) was then used to purify rutin and quercitrin from the switchgrass water extract, which were then tested via the TBARS assay and shown to exhibit lipid peroxidation inhibition activity similar to that obtained with pure flavonoid standards. This is the first report on the presence of rutin and quercitrin in switchgrass. The results support the extraction of viable coproducts from switchgrass prior to conversion to liquid fuel.

Comparison of Two ESI MS Based H/D Exchange Methods for Extracting Protein Folding Energies.

In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate ΔG(H(2)O) and m(GdHCl) values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained ΔG(H(2)O) and m(GdHCl) values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear extrapolation method (LEM) plots is not a reliable indicator of the validity of the data obtained using the LEM approach.

Lipid Compositions in Escherichia coli and Bacillus subtilis During Growth as Determined by MALDI-TOF and TOF/TOF Mass Spectrometry.

Lipids in Escherichia coli and Bacillus subtilis were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and TOF/TOF tandem mass spectrometry. Lipids were extracted from bacterial cells using an equal volume mixture of dichloromethane, ethanol, and water, which formed a biphasic system with the lipids in the organic layer. The resulting mass spectra of the extracts from both bacteria showed a series of peaks corresponding to sodiated phospholipids - primarily phosphatidylethanolamines (PE) and phosphatidylglycerols (PG). The relative amounts of the phospholipids and the fatty acid compositions inferred from the spectra were in good agreement with previously reported values from GC/MS and thin-layer chromatography studies. E. coli and B. subtilis were easily differentiated by dissimilarities in the composition and relative amounts of the phospholipids present as well as by the presence of lysyl-phosphatidylglycerol and diglucosyl diglycerides solely in the B. subtilis mass spectra. Changes in lipid content in the bacteria during their growth phases were also monitored. In E. coli, the spectra indicated an increase in the amount of the unique C(cy-17) fatty acid (in which the fatty acid chain contains a cyclopropane ring) formed during exponential growth. During stationary growth, the spectra indicated an increase in the amount of saturated fatty acids. In B. subtilis, the phospholipid composition remained relatively unchanged during exponential growth, but the amount of PG slightly decreased while the amount of PE slightly increased during stationary growth. No significant changes were observed for the lysyl-phosphatidylglycerols or glycolipids during the exponential or stationary growth phases.

Reducing fragmentation observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of triacylglycerols in vegetable oils.

Edible oils consist primarily of triacylglycerols (TAGs). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of the oils are typically dominated by sodium adducts of these TAGs but also show prominent fragment ions (that do not contain sodium), which can interfere with analytical measurements of other components in oils. The fragments seemingly correspond to the loss of a fatty acid moiety from the sodiated TAGs as a sodium salt: RCOONa. However, a previous study suggested that the fragments actually arise from nearly complete fragmentation of unseen protonated TAGs. These authors suggested that the fragmentation occurs so rapidly and completely that protonated TAGs are not normally observed in the spectra of these oils. In this paper, we present evidence to support their theory and also demonstrate an approach to eliminate these interfering ions from the MALDI-TOF mass spectra via addition of a base to the matrix/sample mixture. The added base does not impede formation of the sodiated TAGs, but does significantly reduce the amount of fragments observed. We propose that this occurs by depleting the H+ ions from the matrix, thus preventing the formation of significant numbers of protonated TAGs in the first place. For measurements by MALDI-TOF, the relative abundances of the fragment ions are related to the strength of the base, and can be almost completely eliminated. However, in longer time-scale experiments such as in post-source decay and Fourier transform mass spectrometry, sodiated and non-sodiated diacylglycerol (DAG)-like fragments are present in spectra, regardless of whether or not a base is added to the sample.

Structural analysis of metal interactions with the dinucleotide duplex, dCG x dCG, using ion mobility mass spectrometry.

The metal binding properties of the dinucleotide duplex, dCG x dCG, were analyzed in the gas phase with ion mobility mass spectrometry. Both MALDI and ESI were used to generate [M(dCG x dCG)]+ complexes. The collision cross section of each complex was measured in helium using ion mobility based methods and compared to calculated cross sections of theoretical structures. When metal cations classified as hard acids were combined with dCG x dCG, the [M(dCG x dCG)]+ complex organized into a globular structure. However, when soft acid metal cations were examined, a structure was observed where the two C-G base pairs were Watson-Crick bound.

Duplex formation and the onset of helicity in poly d(CG)n oligonucleotides in a solvent-free environment.

The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale.

Diastereomer assignment of an olefin-linked bis-paracyclophane by ion mobility mass spectrometry.

trans-1,2-Bis([2.2]paracyclophanyl)ethene (1) exists as a pair of diastereomers whose conformations, and thus effective collision cross sections, are quite different. The two forms can be obtained by different transition metal-catalyzed reactions. To assign meso and racemic structures, a novel method is reported in which experimental gas-phase ion mobility data are compared with theoretical structures obtained from molecular mechanics calculations.

Gas-phase conformations of deprotonated trinucleotides (dGTT-, dTGT-, and dTTG-): the question of zwitterion formation.

The gas-phase conformations of a series of trinucleotides containing thymine (T) and guanine (G) bases were investigated for the possibility of zwitterion formation. Deprotonated dGTT-, dTGT-, and dTTG- ions were formed by MALDI and their collision cross-sections in helium measured by ion mobility based methods. dTGT- was theoretically modeled assuming a zwitterionic and non-zwitterionic structure while dGTT- and dTTG- were considered "control groups" and modeled only as non-zwitterions. In the zwitterion, G is protonated at the N7 site and the two neighboring phosphates are deprotonated. In the non-zwitterion, G is not protonated and only one phosphate group is deprotonated. Two conformers, whose cross-sections differ by 17 +/- 2 A2, are observed for dTGT- in the 80 K experiments. Multiple conformers are also observed for dGTT- and dTTG- at 80 K, though relative cross-section differences between the conformers could not be accurately obtained. At higher temperatures (>200 K), the conformers rapidly interconvert on the experimental time scale and a single "time-averaged" conformer is observed in the ion mobility data. Theory predicts only one low-energy conformation for the zwitterionic form of dTGT- with a cross-section 8% smaller than experimental values. Additionally, the extra H+ on G does not bridge both phosphates. Thus, dTGT- does not appear to be a stable zwitterion in the gas-phase. Theory does, however, predict two low-energy conformers for the non-zwitterionic form of dTGT- that differ in cross-section by 18 +/- 3 A2, in good agreement with the experiment. In the smaller cross-section form (folded conformer), G and one of the T bases are stacked while the other T folds towards the stacked pair and hydrogen bonds to G. In the larger cross-section form (open conformer), the unstacked T extends away from the T/G stacked pair. Similar folded and open conformers are predicted for all three trinucleotides, regardless of which phosphate is deprotonated.

Gas-phase conformations of cationized poly(styrene) oligomers.

The gas-phase conformations of poly(styrene) oligomers cationized by Li+, Na+, Cu+, and Ag+ (M+ PSn) were examined using ion mobility experiments and molecular mechanics/ dynamics calculations. M+PSn ions were formed by MALDI and their ion-He collision cross-sections were measured by ion mobility methods. The experimental collision cross-sections of each M+PS n-mer were similar for all four metal cations and increased linearly with n. Molecular modeling of selected M+ PS oligomers cationized by Li+ and Na+ yielded quasi-linear structures with the metal cation sandwiched between two phenyl groups. The relative energies of the structures were approximately 2-3 kcal/mol more stable when the metal cation was sandwiched near the middle of the oligomer chain than when it was near the ends of the oligomer. The cross-sections of these theoretical structures agree well with the experimental values with deviations typically around 1-2%. The calculations also show that the metal cation tends to align the phenyl groups on the same side of the -CH2-CH- backbone. Calculations on neutral poly(styrene), on the other hand, showed structures in which the phenyl groups were more randomly positioned about the oligomer backbone. The conformations and metal-oligomer binding energies of M+PS are also used to help explain CID product distributions and fragmentation mechanisms of cationized PS oligomers. etry