Hybrid Linear Ion Trap-orbitrap Mass Research Articles

A comprehensive strategy based on ultra-high performance liquid chromatography with diode array detector fingerprinting and multi-component ultra-performance liquid chromatography with tandem mass spectrometry technology for quality control of Jiawei Huoxiang Zhengqi Pill.

Jiawei Huoxiang Zhengqi Pill (JHZP) is a commonly used Chinese patent medicine for the clinical treatment of headache, dizziness, chest tightness as well as abdominal distension, and pain caused by wind-cold flu. In this study, a comprehensive strategy combining ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD) fingerprinting and multi-component quantitative analysis was established and validated for quality evaluation of JHZP. A total of 49 characteristic common peaks were selected in a chromatographic fingerprinting study to assess the similarity of 15 batches of JHZP. Furthermore, 109 compounds were identified or preliminarily identified from JHZP by coupling with an advanced hybrid linear ion trap-Orbitrap mass spectrometer. For quantification, the optimized ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was employed for the simultaneous determination of 13 target compounds within 12min. The sensitivity, precision, reproducibility, and accuracy of the method were satisfactory. This validated UPLC-MS/MS method was successfully applied to analyzing 15 batches of JHZP. The proposed comprehensive strategy combining UHPLC-DAD fingerprinting and multi-component UPLC-MS/MS analysis proved to be highly efficient, accurate, and reliable for the quality evaluation of JHZP, which can be considered as a reference for the overall quality evaluation of other Chinese herbal formulations.

Comprehensive online two-dimensional liquid chromatography coupled to hybrid linear ion trap Orbitrap mass spectrometry with optimized dilution and modulation for analysis of Huangqi Guizhi Wuwu decoction.

Huangqi Guizhi Wuwu decoction (HGWD), as a classical formula, has been used for thousands of years in China. In this work, a comprehensive strategy was proposed for characterizing the chemical profile of HGWD based on online two-dimensional hydrophilic interaction and reversed-phase liquid chromatography coupled with hybrid linear ion trap-Orbitrap mass spectrometry (online HILIC × RP-ESI/HRMS/MSn). The compounds in HGWD were first separated by the combined use of an XBridge amide column (150 × 4.6mm, 3.5μm) and Accucore C18 column (50mm × 4.6mm, 2.6μm). Modulation with assistant technology, including trap columns and online dilution, was optimized and developed to decrease potential analyte loss and improve the resolution of the system. Subsequently, the accurate mass was determined by high-resolution Orbitrap and MSn fragment data by a hybrid linear ion trap (LTQ). In total, 170 chemical constituents were unambiguously identified or tentatively characterized in both positive and negative ion modes. Our study demonstrated that the proposed online HILIC × RP system coupled to the LTQ-Orbitrap MS platform is an efficient analytical technique for characterizing the chemical profile of multicomponent systems.

Protective Effects of Arbutus unedo L. Honey in the Alleviation of Irinotecan-Induced Cytogenetic Damage in Human Lymphocytes-An In Vitro Study.

Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

Protective role of strawberry tree (Arbutus unedo L.) honey against cyto/genotoxic effects induced by ultraviolet B radiation in vitro

Strawberry tree (Arbutus unedo L.) honey (STH) is widely used as part of traditional medicine in the Mediterranean area, especially in the treatment of wounds, burns and infections. In this study, we investigated the cyto-/genoprotective properties of STH at cell level following short-term exposure to UVB radiation at 2 kJ/m2. Isolated human peripheral blood lymphocytes (PBL) were selected as model cells. Phenolics in STH were determined using an ultra-high-performance liquid chromatograph (UHPLC) coupled to a linear ion trap-Orbitrap hybrid mass spectrometer (LTQ Orbitrap MS), while homogentisic acid (HGA) was quantified using a gas chromatograph-mass spectrometer (GC-MS). Primary DNA damage in PBLs treated with STH before and after irradiation was assessed by alkaline comet assay, while their viability was determined using the apoptosis/necrosis assay. The levels of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) were determined in plasma samples. STH applied as pre-treatment at a concentration equivalent to its average daily portion efficiently counteracted the cytotoxic and genotoxic effects and diminished UVB-induced oxidative stress. Post-radiation treatment with STH was not as beneficial. Protective effects could be associated to the complex phytochemical profile of STH and the high content of HGA (306.8 mg/kg). It was confirmed that an intake of a complex mixture of bioactive constituents as STH prior to potential cyto/genotoxic insults stimulated a broad-spectrum of protective mechanisms, including intracellular antioxidative defence and DNA repair systems that helped pre-treated cells to counteract harmful exposures more efficiently.

Chemical characterization of flavonoids and alkaloids in safflower (Carthamus tinctorius L.) by comprehensive two-dimensional hydrophilic interaction chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry

Safflower (Carthamus tinctorius L.) is a famous food additive and herbal medicine in China. In the present research, an online comprehensive two-dimensional hydrophilic interaction chromatography coupled to a diode array detector and a hybrid linear ion trap-Orbitrap mass spectrometry (HILIC × HILIC-DAD-ESI/HRMS/MSn) platform was developed to analyze the flavonoids and alkaloids in safflower. By combining with an XBridge Amide column (150 mm × 4.6 mm, 3.5 μm) and an Ultimate amide column (50 mm × 4.6 mm, 5 μm), the system orthogonality reached 88% and a total of 231 peaks were detected. Altogether 93 compounds, including 75 flavonoids and their glycosides and 10 alkaloids were unambiguously or tentatively identified in both negative and positive ion modes, using accurate mass and MS fragment data. Among them, 5 compounds were discovered and reported from safflower for the first time. The established HILIC × HILIC platform should be a powerful tool for the separation and characterization of complicated matrices in natural herbs.

Yinchenzhufu decoction protects against alpha-naphthylisothiocyanate-induced acute cholestatic liver injury in mice by ameliorating disordered bile acid homeostasis and inhibiting inflammatory responses

Ethnopharmacological relevanceIntrahepatic cholestasis is a common condition of many liver diseases with few therapies. Yinchenzhufu decoction (YCZFD) is a representative traditional Chinese herbal formula used for treating jaundice and liver disease. Aim of the studyTo investigate the hepatoprotective effect of YCZFD against cholestatic liver injury and reveal its potential mechanism. Materials and methodsMice with alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis were orally administered YCZFD at doses of 3, 6, and 12g crude drug/kg for 2 weeks followed by subsequent analyses. A serum metabolomics study was then performed to explore the different metabolites influenced by YCZFD using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap hybrid mass spectrometry (UPLC-LTQ-Orbitrap-MS/MS).The levels of individual bile acids in the serum, liver, and bile were determined by UPLC-MS/MS. The expression of metabolic enzymes, transporters, inflammatory factors, and cytokeratin-19 (CK-19) was determined by real-time PCR, western blotting, and immunohistochemistry. ResultsYCZFD administration decreased the serum biochemical indexes and ameliorated pathological damage, such as hepatic necrosis and inflammatory cell infiltration. Serum metabolomics revealed that the metabolites influenced by YCZFD were mainly associated with bile acid metabolism and inflammation. YCZFD administration effectively ameliorated the disordered bile acid homeostasis. The bile acid transporter, multidrug-resistance associated protein 2 (Mrp2), and the metabolic enzyme, cytochrome P450 2b10 (Cyp2b10), were upregulated in the YCZFD intervention group compared to those in the ANIT-induced group. YCZFD administration also significantly inhibited nuclear factor-κB (NF-κB) and its phosphorylation and decreased the expression of proinflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and intercellular adhesion molecule-1 in ANIT-induced cholestatic mice. Additionally, the level of CK-19 was lower in the YCZFD intervention group than in the ANIT-induced cholestatic mice. ConclusionYCZFD administration ameliorated disordered bile acid homeostasis, inhibited NF-κB pathway-mediated inflammation, and protected the liver from bile duct injury. Therefore, YCZFD exerted a protective effect against cholestatic liver injury.

Chemometric Characterization of Strawberries and Blueberries according to Their Phenolic Profile: Combined Effect of Cultivar and Cultivation System.

Chemical characterizations of leaves and fruits that were obtained from organically and integrally produced strawberries (′Favette′, ′Alba′, and ′Clery′) and blueberries (′Bluecrop′, ′Duke′, and ′Nui′) from western Serbia were undertaken in this study. Phenolic analysis was done while using ultra-high performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass analyzer, while total phenolic content (TPC), total anthocyanin content (TAC), and radical-scavenging activity (RSA) by spectrophotometry. In general, leaves and fruits from blueberry showed higher levels of TPC and TAC as compared to strawberry. These chemical traits were larger in organic grown fruits and larger in leaves than fruits. The most abundant phenolics in leaves and fruits of blueberry was 5-O-caffeoylquinic acid, followed by quercetin 3-O-galactoside, while catechin, quercetin, and kaempferol 3-O-glucosid were dominant in the leaves and fruits of strawberry. cis, trans-Abscisic acid was detected in all fruit samples, but not in leaves. Blueberries (both fruits and leaves) were separated from strawberries, but only organic blueberry fruits were distinguished from integrated fruits, according to principal component analysis. Quercetin, kaempferol, 5-O-caffeoylquinic acid, ferulic acid, caffeic acid, catechin, p-coumaric acid, and p-hydroxybenzoic acid were the most influential phenolic compounds for the separation. Much higher contents of TPC, RSA, TAC, quercetin 3-O-galactoside, and quercetin were found in fruits and TPC, RSA, catechin, p-hydroxybenzoicacid, p-coumaricacid, and ferulic acid in leaves in all three blueberry cultivars and the strawberry cultivar ′Clery′. These phenolic compounds are good sources of antioxidant compounds with potentially high beneficial effects on human health.

Comprehensively qualitative and quantitative analysis of ginsenosides in Panax notoginseng leaves by online two-dimensional liquid chromatography coupled to hybrid linear ion trap Orbitrap mass spectrometry with deeply optimized dilution and modulation system

Panax notoginseng leaves (PNL) was considered as a potential medicinal part with abundant protopanaxdiol type ginsenosides. In this study, an integrated system was developed for simultaneously qualitative and quantitative analysis of ginsenosides in PNL using online comprehensive two-dimensional hydrophilic interaction chromatography and reversed-phase liquid chromatography coupled to a hybrid linear ion trap-Orbitrap mass spectrometry (online HILIC × RP-ESI/HRMS/MSn). The system was configured based on the combination of a XBridge amide column (150 mm × 2.1 mm, 2.5 μm) and Accucore phenyl-hexyl (50 mm × 4.6 mm, 2.6 μm) for the first and second dimensions, respectively. An additional water phase was introduced to dilute the eluent from the first dimension to decrease its elution strength in the second dimension. The online dilution, modulation interface and the second-dimension gradient program were deeply optimized to reduce possible sample loss and improve system resolution. Under the optimal conditions, a total of 226 ginsenosides were unambiguously identified or tentatively characterized by aid of high-resolution accurate mass and MSn fragment data in both negative and positive ion modes, and 93 of them were discovered as potentially new ginsenosides in PNL. Besides, the validated online HILIC × RP-LTQ-MS method was applied to determine 24 ginsenosides directly on 2D-EIC contour plots in nine batches of PNL samples. The powerful separation capability acquired by the developed online HILIC × RP system affords not only reliable structural information for identification, but also accurate quantitation. This combined system can also be used to characterize and quantify bioactive ingredients in the samples with complex matrices.

Identification of polyphenolic composition in the fruits of Rubus chingii Hu and its antioxidant and antiproliferative activity on human bladder cancer T24 cells

Rubus chingii Hu, namely “Fu-pen-zi” in Chinese, is commonly used as a functional food in China. A rapid and sensitive High performance liquid chromatography (HPLC) coupled with linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrap MS) was employed to identify the polyphenolic compounds in R. chingii fruits. A total of 18 compounds were identified in R. chingii polyphenols (RCPs), including five phenolic acids, four ellagitannins, and nine flavonoids. Additionally, the total phenolics, total flavonoids and total tannins content as well as antioxidant and antiproliferative activity were evaluated for the first time. The contents of total phenolics, total flavonoids, and total tannins were 356.42 mg GAE g−1 of extract, 201.94 mg RUE g−1 of extract and 155.12 mg GAE g−1 of extract, respectively. Antioxidant assays showed RCPs had favorable DPPH and OH− radical scavenging capacity and strong ferric ion reducing power. Moreover, the results also indicated the RCPs could inhibit cell proliferation and induce the cell apoptosis in T24 bladder cancer cells. Altogether, the results suggest R. chingii possesses potent antioxidant properties and could inhibit the growth of T24 cells by inducing the cell apoptosis in a dose-dependent manner.

Anti-quorum sensing activity, toxicity in zebrafish (Danio rerio) embryos and phytochemical characterization of Trapa natans leaf extracts

Ethnopharmacological relevanceTrapa natans L. (water chestnut or water caltrop) is a widespread aquatic plant, which has been cultivated for food and traditional medicine since ancient times. Pharmacological studies showed that water chestnut exhibits the wide range of biological activities, such as antimicrobial, antioxidative, analgesic, anti-inflammatory, as well as antiulcer. Aim of the studyEvaluation of anti-virulence potential and toxicity of T. natans methanol (TnM), acetone (TnA) and ethyl acetate (TnEA) leaf extracts. Materials and methodsThe anti-quorum sensing activity of Tn extracts was addressed by measuring their effects on biofilm formation, swarming motility and pyocyanin and elastase production in Pseudomonas aeruginosa. Specific P. aeruginosa biosensors were used to identify which of the signaling pathways were affected. The lethal and developmental toxicity of extracts were addressed in vivo using the zebrafish (Danio rerio) model system. The phenolic composition of T. natans leafs extracts was analyzed by a linear ion trap-OrbiTrap hybrid mass spectrometer (LTQ OrbiTrapMS) and UHPLC system configured with a diode array detector (DAD) hyphenated with the triple quadrupole mass spectrometer. ResultsSubinhibitory concentrations of Tn leaf extracts (0.2 MIC) inhibited pyocyanin and elastase production up to 50% and 60%, respectively, and reduced swarming zones, comparing to non-treated P. aeruginosa. TnA inhibited biofilm formation by 15%, TnM showed a stimulatory effect on biofilm formation up to 20%, while TnEA showed no effect. The bioactive concentrations of TnM and TnA were not toxic in the zebrafish model system. Twenty-two phenolic compounds were tentatively identified in TnM, where thirteen of them were identified in T. natans for the first time. Tn extracts, as well as their major components, ellagic and ferulic acids, demonstrated the ability to interfere with P. aeruginosa Las and PQS signaling pathways. ConclusionsThis study demonstrates anti-virulence potential of Tn leaf extracts against medically important pathogen P. aeruginosa and confirms the ethnopharmacological application of this plant against microbial infections.

The Application of Ultra-High-Performance Liquid Chromatography Coupled with a LTQ-Orbitrap Mass Technique to Reveal the Dynamic Accumulation of Secondary Metabolites in Licorice under ABA Stress.

The traditional medicine licorice is the most widely consumed herbal product in the world. Although much research work on studying the changes in the active compounds of licorice has been reported, there are still many areas, such as the dynamic accumulation of secondary metabolites in licorice, that need to be further studied. In this study, the secondary metabolites from licorice under two different methods of stress were investigated by ultra-high-performance liquid chromatography coupled with hybrid linear ion trap–Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS). A complex continuous coordination of flavonoids and triterpenoids in a network was modulated by different methods of stress during growth. The results showed that a total of 51 secondary metabolites were identified in licorice under ABA stress. The partial least squares–discriminate analysis (PLS-DA) revealed the distinction of obvious compounds among stress-specific districts relative to ABA stress. The targeted results showed that there were significant differences in the accumulation patterns of the deeply targeted 41 flavonoids and 10 triterpenoids compounds by PCA and PLS-DA analyses. To survey the effects of flavonoid and triterpenoid metabolism under ABA stress, we inspected the stress-specific metabolic changes. Our study testified that the majority of flavonoids and triterpenoids were elevated in licorice under ABA stress, while the signature metabolite affecting the dynamic accumulation of secondary metabolites was detected. Taken together, our results suggest that ABA-specific metabolite profiling dynamically changed in terms of the biosynthesis of flavonoids and triterpenoids, which may offer new trains of thought on the regular pattern of dynamic accumulation of secondary metabolites in licorice at the metabolite level. Our results also provide a reference for clinical applications and directional planting and licorice breeding.

Online comprehensive two-dimensional hydrophilic interaction chromatography × reversed-phase liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry for the analysis of phenolic acids in Salvia miltiorrhiza

In the present study, a system was developed for the analysis of phenolic acids in Salvia miltiorrhiza using online comprehensive two-dimensional hydrophilic interaction chromatography and reversed-phase liquid chromatography coupled to a DAD detector and hybrid linear ion trap-Orbitrap mass spectrometry (HILIC×RP-DAD-ESI/HRMS/MSn). The system was configured based on the combination of an XBridge Amide column (150mm×4.6mm, 3.5μm) and Accucore PFP column (50mm×4.6mm, 2.6μm) for the first and second dimensions, respectively. An additional LC pump was used to dilute the eluent from the first dimension to decrease its elution strength in the second dimension. A back-flush trap column was selected as an interface to make up for the loss of efficiency and resolution due to the online dilution. Under the optimized conditions, a total of 196 peaks of polar compounds were successfully separated and detected in Salvia miltiorrhiza using the developed online HILIC×RP system, which exhibited high orthogonality (73%). The online combination of HILIC and RP provides powerful separation capability for the analysis of polar compounds in samples with complex matrices.

Application of HPLC-LTQ Orbitrap MS for metabolic profiles of Polygonum multiflora extract in rats.

The root of Polygonum multiflorum (PM) is an important Chinese herbal medicine for treatment of various diseases. Extensive pharmacological studies have been conducted and demonstrated that it shows a wide range of bioactivities. Meanwhile, a considerable number of hepatotoxicity cases owing to oral administration of PM have been reported and have attracted great attention. However, the limited knowledge regarding the metabolism of PM restricts the deeper studies on its pharmacological/toxicological mechanism and therapeutic material basis. The present study aimed to develop a high-performance liquid chromatography coupled with a linear ion trap-Orbitrap hybrid mass spectrometry method for separation and identification of metabolites in rat urine and plasma after oral administration of PM. Based on the proposed strategy, metabolism profiles of PM in rats were proposed for the first time and 43 metabolites were characterized or tentatively identified. Phase II metabolism, such as glucuronidation and sulfation, are the principal pathways of the main components. These findings will be beneficial to further understanding of the pharmacological mechanism and pharmacodynamic material basis of PM.

Flexible and powerful strategy for qualitative and quantitative analysis of valepotriates in Valeriana jatamansi Jones using high-performance liquid chromatography with linear ion trap Orbitrap mass spectrometry.

Valeriana jatamansi Jones is an important medicinal plant and its quality is closely related to its region of origin. In the current study, we utilized a flexible and powerful strategy for comprehensiveevaluation of the quality diversity for 15 regions in China. The method was based on a hybrid linear ion trap-Orbitrap mass spectrometry platform. For structure characterization,fragmentation patterns were detected by analyzing a series of standard compounds using data dependent multistage mass spectrometry acquisition. A fragment ion database for valepotriates was established, and the acquired data were high throughput filtered by fragment ion search for compound identification. For quantitative purposes, we normalized the mass spectrometry data of 15 samples using SIEVE 2.0 and the differences in composition were analyzed using principal component analysis combined with hierarchical clustering analysis. The results identified a total of 92 compounds fromValeriana jatamansi Jones. Samples from Dali, Kunming, and Baoshan have better qualities and concentrations of the main activeconstituents. To verify our strategy, we compared the valtrate, acevaltrate, and baldrinal contents using high-performance liquid chromatography with diode array detector. We developed and validated a comprehensive qualitative and quantitative analytical method to achieve quality control of Valeriana jatamansi Jones.

Assessment of Photodegradation of Herbicide Prometryn in Soil

Prometryn has been used in crop (e.g., corn and sorghum) field to prevent growth of annual grasses and broadleaf weeds for many years. As a moderately persistent herbicide in soil, prometryn may exert detrimental effects on environmental safety and crop production. The present study assessed the photodegradation of prometryn residues in soil by exploring a variety of factors such as soil moisture, temperature, and light exposure that potentially affect prometryn photodegradation. The dissipation rate of prometryn during a 14-day period of study was more than 90% under 15 (low pressure), 100, and 300 W (medium pressure) UV light exposure. The half-life of prometryn decay under UV light (53.5–116.4 h) was far less than that under xenon light (1131.6 h) and dark (3138.7 h) conditions. When the soil moisture (clay loam) was 60% of the field moisture capacity, it was most effective for prometryn photodegradation. The prometryn photodegradation on soil with 60% moisture level was increased with temperature and prometryn concentrations. The theoretical optimization scheme for eliminating prometryn in soil was recommended. The degraded products of prometryn under UV light and darkness were characterized using ultra high-performance liquid chromatography coupled to a linear ion trap-orbitrap hybrid mass spectrometer (UPLC-LTQ-orbitrap-MS/MS) and showed that prometryn decay in soil was through hydroxylation, dealkylation, and dethiomethylation pathways.

Ultra-high performance liquid chromatography with linear ion trap-Orbitrap hybrid mass spectrometry combined with a systematic strategy based on fragment ions for the rapid separation and characterization of components in Stellera chamaejasme extracts.

Stellera chamaejasme, a famous toxic herb, has been used in traditional Chinese medicine to treat various diseases. For decades, increasing attention in modern pharmacological studies has been drawn to S. chamaejasme because of its potential anti-tumor, anti-virus, and anti-fungus activities. However, due to the intrinsic complexity of chemical constitutes, hardly any investigations formed an overall recognition for the chemical profiles of this herb. In this study, a rapid and sensitive ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry method was developed to characterize the chemical components of S. chamaejasme extracts. Based on optimized ultra-high performance liquid chromatography and mass spectrometry conditions and systematic fragment ions-based strategy, a total of 47 components including flavones, diterpenes, coumarins, and lignans were simultaneously detected and identified or tentatively identified for the first time. The MS(n) fragmentation patterns of all the characterized compounds in positive or negative electrospray ionization modes were also explored and summarized. These results provided essential data for further pharmacological research on S. chamaejasme. Moreover, the method was demonstrated to be an efficient tool for rapid qualitative analysis of secondary metabolites from natural resources.

Monitoring of twenty-two sulfonamides in edible tissues: Investigation of new metabolites and their potential toxicity

The extensive and unregulated use of antibacterial drugs in animal farms in Lebanon can lead to detrimental consequences for the public health. To monitor the levels of sulfonamides and their metabolites in farms in Lebanon, a total of 304 meat samples were collected and analyzed using liquid chromatography coupled to triple quadrupole and hybrid linear ion trap-Orbitrap mass spectrometry following QuEChERS-based extraction. Sulfonamide residues could be detected in forty-six samples, ten of which contained a concentration of sulfaquinoxaline (151.4–1196.7μgkg−1 in chicken samples) and sulfadiazine (109.8μgkg−1 in a beef sample) exceeding the European Union-based maximum residue level by 1–12 folds, and thus were unfit for human consumption. Several acetylated, hydroxylated, and/or sulfated metabolites were identified, some of which were not previously detected in edible tissues. Most identified metabolites exhibited potential toxicity equivalent or higher than that of the parent molecule as estimated by in silico tests.

Uncovering protein polyamination by the spermine-specific antiserum and mass spectrometric analysis.

The polyamines spermidine and spermine, and their precursor putrescine, have been shown to play an important role in cell migration, proliferation, and differentiation. Because of their polycationic property, polyamines are traditionally thought to be involved in DNA replication, gene expression, and protein translation. However, polyamines can also be covalently conjugated to proteins by transglutaminase 2 (TG2). This modification leads to an increase in positive charge in the polyamine-incorporated region which significantly alters the structure of proteins. It is anticipated that protein polyamine conjugation may affect the protein-protein interaction, protein localization, and protein function of the TG2 substrates. In order to investigate the roles of polyamine modification, we synthesized a spermine-conjugated antigen and generated an antiserum against spermine. In vitro TG2-catalyzed spermine incorporation assays were carried out to show that actin, tubulins, heat shock protein 70 and five types of histone proteins were modified with spermine, and modification sites were also identified by liquid chromatography and linear ion trap-orbitrap hybrid mass spectrometry. Subsequent mass spectrometry-based shotgun proteomic analysis also identified 254 polyaminated sites in 233 proteins from the HeLa cell lysate catalyzed by human TG2 with spermine, thus allowing, for the first time, a global appraisal of site-specific protein polyamination. Global analysis of mouse tissues showed that this modification really exists in vivo. Importantly, we have demonstrated that there is a new histone modification, polyamination, in cells. However, the functional significance of histone polyamination demands further investigations.

Chromatogram-structure analysis: An untargeted workflow for holistic visualizing chemical composition in macrocyclic diterpene esters based on ultra high performance Liquid Chromatography Hybrid Linear Ion Trap–Orbitrap Mass Spectrometry

Chromatogram-structure analysis: An untargeted workflow for holistic visualizing chemical composition in macrocyclic diterpene esters based on ultra high performance Liquid Chromatography Hybrid Linear Ion Trap–Orbitrap Mass Spectrometry

The fronds tonoplast quantitative proteomic analysis in arsenic hyperaccumulator Pteris vittata L.

Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1mM arsenate for 3weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography-a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an "artificial" database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata. Vacuole compartmentalization is crucial to As hyperaccumulator P. vittata, while there is limited known arsenic transport proteins involved in vacuole compartmentalization. In this paper, we obtained tonoplast of P. vittata fronds by iodixanol step gradient centrifugation method and then used TMPP protein labeling proteome technology for the quantitative detection of fronds tonoplast proteins. Our findings are the first challenge to the tonoplast proteins data mining of P. vittata which provide an informative basis for As carriers or channels. The proteomic approach in our study is suited for detecting alterations tonoplast protein and help to clarify the regulation of tonoplast transport processes. This article is part of a Special Issue entitled: Proteomics of non-model organisms.