Ultrahigh Performance Liquid Chromatography-quadrupole/time Of Flight Research Articles

Can postmortem metabolomics be used as a tool in the identification of deaths attributed to hypoglycaemia

To establish a metabolomic profile that could be used as a screening tool for possible deaths attributed to hypoglycaemia. Postmortem metabolomics can be used to assist death investigations by characterising new biomarker profiles differentiating causes of death. Hypoglycaemia-related deaths, particularly insulin intoxications, are difficult to identify without support from antemortem blood samples. The biological analysis of exogenous insulins in postmortem cases is troublesome, as immunoassays are not precise when handling haemolysed blood samples from postmortem cases. However, in recent years advances have been made in identification of insulin analogues, particularly in vitreous fluid. Nevertheless, due to such limitations it is probable that a proportion of hypoglycaemia-related deaths remain unknown. The aim of this investigation is to build a postmortem metabolomics model to be used as a tool to identify deaths related to hypoglycaemia. Data from ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry was obtained from postmortem cases including; 19 insulin intoxication cases (hypoglycaemia group), 19 diabetic coma cases (hyperglycaemia group), and 38 hanging cases (control group). Additionally, a random test group of postmortem cases ( n = 726) were used for test screening for possible unidentified hypoglycaemic deaths. Mass spectrometry data was processed in XCMS using R. Orthogonal-partial least squares discriminant analysis (OPLS-DA), in SIMCA, was used to build models comparing metabolomic features between hypoglycaemia and control groups, and hypoglycaemia and hyperglycaemia groups. These two models were combined into a shared-unique-structures (SUS) plot, and the discriminating features specific for hypoglycaemia group were selected for identification and used to build the screening model. Features were identified using the HMDB and METLIN databases. XCMS data analyses resulted in the identification of 1775 metabolomic features. The OPLS-DA model for hypoglycaemia vs control resulted in complete group separation with R2 = 0.90 and Q2 = 0.62, and the model for hypoglycaemia vs hyperglycaemia resulted in complete group separation with R2 = 0.93 and Q2 = 0.86. The SUS plot of these two models resulted in 83 features that were considered to discriminate the hypoglycaemia group. An OPLS-DA model using only those 83 discriminating features was built, resulting in complete group separation of hypoglycaemia, hyperglycaemia, and control groups (R2 = 0.73, Q2 = 0.56). Group classification prediction was performed for the random test group on this model, which resulted in 46 (6.0%), 17 (2.2%), and 332 (43.5%) of cases being classified as belonging to hypoglycaemia, hyperglycaemia, and control groups, respectively. Cases classified as belonging to no group were 363 (47.6%), falling below the threshold of group classification in the model, and the remaining four cases were classified as belonging to two of the three groups in various combinations. The reported causes of death for those classified as hypoglycaemia showed a varied list of causes, including cardiovascular complications, substance overdose/poisoning, asphyxia, amongst others. However, six cases have an undetectable cause of death reported. The difficulties in the identification of hypoglycaemia-related deaths and the toxicological analysis of exogenous insulin remains. However, based on the results here we can see that postmortem metabolomics is able to discriminant deaths attributed to hypoglycaemia, hyperglycaemia, and non-glycaemic deaths (controls). Additionally, the model for postmortem case screening classified a discrete group of the random test group (only 6%) as hypoglycaemia, and whilst the individual cases still require review, this low number of classifications is a good indication that a viable screening model could be developed. Postmortem metabolomics is a useful tool in the screening of possible hypoglycaemia-related deaths. Further studies are required to expand on the potential of postmortem metabolomics as a useful tool in the investigation of hypoglycaemia-related deaths, and the future application of identifying potential insulin intoxications.

Characterization of 17 unknown ketamine manufacturing by-product impurities by UHPLC-QTOF-MS.

This study initially reported the selection and characterization of 17 unknown impurities attributed to the manufacture process of ketamine. A total of 150 seized ketamine samples were investigated through ultra-high-performance liquid chromatography-quadrupole-time of flight (UHPLC-Q-TOF). Seventeen characteristic impurities were selected in accordance with four criteria: The compound was detected in over 10% of all 150 seized ketamine samples, the compound had at least one nitrogen, the unsaturation of the compound was more than 5, and the compound was stable in the dilution solvent solution for 48h. The accurate masses of the protonated molecules and product ions of the target impurities were obtained based on the full scan mode and the product ion mode of Q-TOF, respectively. Lastly, the possible structures of the above impurities were tentatively elucidated in accordance with the synthetic route of ketamine, protonated molecules, and MS2 product ions. All 17 impurities had the same skeleton of deschloroketamine (DCK), but were substituted with additional chlorine, hydroxyl, methyl, cyclohexane, and o-chlorophenyl cyclopentyl ketone substituents. Under the electrospray ionization (ESI), the above impurities showed similar characteristic fragment ions through the dissociation of the CH3NH2, C2H6NH, H2O, CO, C2H4O, C4H6, and C2H2 moieties. The above impurities have been routinely used for the profiling analysis of seized ketamine samples in the National Narcotics Laboratory of China and employed to establish the tactical intelligence for law enforcement agencies.

Coupling between high-resolution mass spectrometry and focalized data-analysis methods provides the identification of new putative glycosidic non-anthocyanic flavonoids in grape.

The biochemical diversity of flavonoids is based on glycosylation, methylation, acylation, and many other modifications of the flavonoid backbone. Liquid chromatography coupled to high-resolution mass spectrometry demonstrated to be a powerful approach to gain new insights into the flavonoid composition of many plant species, including grapes. Among different metabolomic approaches, suspect screening analysis relies on the construction of a specific database and on ultra-high performance liquid chromatography/quadrupole time-of-flight (UHPLC/QTOF) analysis to find new compounds of oenological interest. A homemade database containing mass data information retrieved from the literature specific for plant flavonoid derivatives (GrapeFlavMet) was constructed. Tandem mass spectrometry analysis of V. vinifera and hybrid grape extracts was performed, and MS/MS fragmentation allowed to assign the putative flavonoid chemical structure to various identification levels, as established by the Metabolomics Standard Initiative. By this approach, putative flavonoid derivatives with different glycosylation and acylation patterns were identified. They include three pentoside derivatives of tetrahydroxy-flavone, tetrahydroxy-flavanone and myricetin isomers, a putative dihydrorhamnetin hexoside derivative, three cinchonain isomers (phenylpropanoid-substituted flavan-3-ols with antidiabetic properties), and two syringetin isomer derivatives (acetyl- and p-coumaroyl-hexoside). Two acetyl-hexoside derivatives of dihydrorhamnetin and pentahydroxy-methoxy-flavanone, and three derivatives of tetrahydroxy-dimethoxy-flavanone (acetyl, p-coumaroyl, and caffeoyl-hexoside) were tentatively annotated. Most of the compounds were identified in grape for the first time, while two putative syringetin derivatives previously proposed in the literature were confirmed. These findings deepen the current knowledge on grape flavonoids, suggesting more connections at the biochemical level.

Optimization Model of Phenolics Encapsulation Conditions for Biofortification in Fatty Acids of Animal Food Products.

The nutritional quality of animal products is strongly related to their fatty acid content and composition. Nowadays, attention is paid to the possibility of producing healthier foods of animal origin by intervening in animal feed. In this field, the use of condensed tannins as dietary supplements in animal nutrition is becoming popular due to their wide range of biological effects related, among others, to their ability to modulate the rumen biohydrogenation and biofortify, through the improvement of the fatty acids profile, the derivate food products. Unfortunately, tannins are characterized by strong astringency and low bioavailability. These disadvantages could be overcome through the microencapsulation in protective matrices. With this in mind, the optimal conditions for microencapsulation of a polyphenolic extract rich in condensed tannins by spray drying using a blend of maltodextrin (MD) and gum Arabic (GA) as shell material were investigated. For this purpose, after the extract characterization, through spectrophotometer assays and ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry, a central composite design (CCD) was employed to investigate the combined effects of core:shell and MD:GA ratio on the microencapsulation process. The results obtained were used to develop second-order polynomial regression models on different responses, namely encapsulation yield, encapsulation efficiency, loading capacity, and tannin content. The formulation characterized by a core:shell ratio of 1.5:5 and MD:GA ratio of 4:6 was selected as the optimized one with a loading capacity of 17.67%, encapsulation efficiency of 76.58%, encapsulation yield of 35.69%, and tannin concentration of 14.46 g/100 g. Moreover, in vitro release under varying pH of the optimized formulation was carried out with results that could improve the use of microencapsulated condensed tannins in animal nutrition for the biofortification of derivates.

The variegated scallop, Mimachlamys varia, undergoes alterations in several of its metabolic pathways under short-term zinc exposure

The variegated scallop (Mimachlamys varia) is a filter feeder bivalve encountered in marine regions of the Atlantic coast. In particular, it is present in the La Rochelle marina (France), where it is used for the biomonitoring of marine pollution, due to its ability to strongly bioaccumulate pollutants. In this semi-closed environment, contamination generated by port activities leads to an accumulation of both organic and metal pollutants. Zinc is one of these pollutants, present at a dose of up to 150 μg.L−1. This study investigated the effects of 48 h zinc exposure upon the metabolic profiles of Mimachlamys varia using UHPLC/QToF (ultra-high performance liquid chromatography-quadrupole time-of-flight) tandem mass spectrometry metabolomics. After acclimation in mesocosms recreating in situ conditions, both controls and exposed with Zn2+ (150 μg.L−1) bivalves were dissected to recover the gills after 48 h and stored at −80 °C before metabolites extraction. UHPLC/QToF tandem mass spectrometry was performed to study metabolite composition of samples. Statistical analysis of results using multivariate techniques showed a good classification between control and exposed groups. Eleven identified metabolites were found to be down-modulated in exposed scallops. These variations could reflect potential zinc effects on several of the biological processes, such as energy metabolism, osmoregulation and defense against oxidative stress. Among the eleven metabolites highlighted, four were reported for the first time in an aquatic organism exposed to Zn. This study demonstrates once again the diversity of interactions between bivalves and metals and the complexity of the physiological response of marine bivalves to pollutants.

Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry and Rapid Evaporative Ionization Mass Spectrometry Were Used to Develop a Lamb Authentication Method: A Preliminary Study.

A untargeted metabolomics approach was proposed in this study based on ultra-high performance liquid chromatography quadrupole time-of-flight (UHPLC-QTOF) and rapid evaporative ionization mass spectrometry (REIMS) to discriminate lamb and mutton meat and investigate their subtle metabolic differences, considering the higher popularity of lamb meat than mutton in the market. Multivariate statistical analysis was performed for data processing in order to distinguish between the two sample types. A total of 42 potential metabolites (20 in positive and 22 in negative ion mode) were defined for UHPLC-QTOF analysis, which provided references for discriminating the two kinds of meat. Furthermore, three potential markers were tentatively identified using LC/MS data against chemical databases. In addition, 14 potential metabolites were putatively identified in negative ion mode using the LipidMaps database. Meanwhile, the data-driven soft independent modeling of class analogy (DD-SIMCA) model was established, which could rapidly differentiate non-pretreated lamb meat and mutton with 92% specificity, rendering REIMS a promising technique for meat identification.

Uncovering proteome variations of differently heat-treated sea cucumber (Apostichopus japonicus) by label-free mass spectrometry

The effects of heat treatment on the proteome of Apostichopus japonicus have been evaluated using label-free quantitative proteomics by ultrahigh performance liquid chromatography-quadrupole/time of flight (UHPLC-Q/TOF) mass spectrometry with sequential window acquisition of all the theoretical fragment ion (SWATH) acquisition mode. Chemometric tools are integrated to reveal proteomic changes by mining the protein quantitation data from fresh and differently heat-treated samples. SWATH allows the quantitation of 548 proteins, of which 24 proteins are significantly sensitive to heat treatment and 13 proteins vary significantly responding to different heat procedures (boiling, steaming, and microwave heating), and 5 of them are sharing proteins. Gene ontology (GO) annotation of the differentiating proteins highlights most of them are relevant to molecular functions. The results can be favorable to evaluate the effects of heat treatment on the nutrition and function of processed sea cucumbers and facilitate the selection of an optimal thermal treatment.

A UHPLC-QTOF-MS screening provides new insights into the phytochemical composition and biological properties of six Consolida species from Turkey

The genus Consolida is one of the most important within the Ranunculaceae family and several members of this genus contain important biologically active compounds, such as phenolics and alkaloids. The present study attempts for the first time to assess the biological properties and phytochemical constituents of six Consolida species (C. glandulosa (Boiss. & A. Huet) Bornm, C. hellospontica (Boiss.) Chater, C. raveyi (Boiss.) Schrödinger, C. regalis (Boiss.) Schrödinger, C. staminosa P.H. Davis & Sorger and C. stenocarpa (P.H. Davis & M. Hossain) P.H. Davis) growing in Turkey. A comprehensive phytochemical profiling of the different species was achieved by using an ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry, targeting polyphenols and diterpene alkaloids. Also, the in vitro antioxidant assays (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), metal chelating and phosphomolybdenum) and enzyme inhibition (cholinesterases, tyrosinase, α-amylase, and α-glucosidase) potentials of the different Consolida methanolic extracts were evaluated. The UHPLC-QTOF profiling revealed 492 compounds, being 236 flavonoids, 93 phenolic acids, 78 tyrosol derivatives, 49 diterpene alkaloids, 29 lignans, and 7 stilbenes. The cumulative phenolic content ranged from 17.65 mg Eq./g for C. staminosa up to 43.04 mg Eq./g for C. glandulosa, whilst the total diterpene alkaloids content was exclusively found in C. glandulosa extracts (3.53 mg Eq./g). Consolida regalis (58. 61 mg trolox equivalent (TE)/g for DPPH and 65.38 mg TE/g for ABTS) and C. stenocarpa (46.81 mg TE/g for DPPH and 78.12 mg TE/g for ABTS) exhibited the highest anti-radical abilities, while the best reducing power values were recorded for C. glandulosa (129.46 mg TE/g for CUPRAC and 77.01 mg TE/g for FRAP) and C. raveyi (124.56 mg TE/g for CUPRAC and 78.36 mg TE/g for FRAP). Also, following the cholinesterases inhibition assays, C. hellospontica and C. glandulosa exhibited the highest inhibitory effects. Regarding tyrosinase inhibitory effects, C. raveyi showed the highest value, being 126.60 mg kojic acid equivalent (KAE)/g, followed by C. glandulosa (125.82 mg KAE/g) and C. stenocarpa (123.09 mg KAE/g). Besides, Consolida staminosa showed potential inhibition against α-amylase, but low glucosidase inhibitory properties. Therefore, the present findings could provide a starting point for further investigations to promote the industrial exploitation of these species with the aim of designing novel phyto-pharmaceuticals.

UPLC-QTOF-MS Method for Identification of Mango Leaf Tea Metabolites

Abstract Objectives The study aim was to identify bioactive compounds in mango leaf tea using a new method - UPLC/Q-TOF-MS. Mango leaf tea is a natural product being investigated to manage type 2 diabetes due to its phytochemical properties. Methods Mango tea was extracted using 80% methanol overnight. The extract was analyzed with ultra-high-performance liquid chromatography-quadrupole time-of-flight (UPLC-Q-TOF) MS system (Waters Corp., Milford, MA) and injected into an Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm, Waters Corp.). The column was equilibrated with distilled water containing 0.1% formic acid (FA) and eluted by a linear gradient of acetonitrile (ACN) containing 0.1% FA at a flow rate of 0.35 mL/min for 10 min and 40 °C of column temperature. Results The eluents were analyzed by A Vion IMS Q-TOF MS (Waters Corp.) with positive and negative electrospray ionization (ESI). The scan range of TOF MS data was 50–1500 m/z, the scan time was 0.2 sec, and the capillary and sampling cone voltages were 3 kV and 40 V, respectively. The desolvation flow rate and temperature were 800 L/h and 400 °C, respectively, and the source temperature was 100 °C. Leucine-enkephalin ([M + H] = 556.2771 Da; [M-H] = 554.2615 Da), used as a lack mass, was infused at a flow rate of 0.35 μL/min and a frequency of 10 s to ensure mass measurement accuracy of the metabolites analyzed by the instrument. Mass spectral data were collected from m/z 50 to 1500 with a scan time of 0.2 s. Collision energy was ramped from 10 to 40 eV for the collection of MS/MS data. The metabolites Mangiferin, quercetin/isoquercetin and iriflophenone 3-beta-glucoside were identified using ChemSpider in negative mode while only mangiferin and quercetin were defined in positive mode with different intensites: Mangiferin (421.07), quercetin/isoquercetin (303.05) and irrflophenone 3- beta- glucoside (407.097) that confirmed their molar mass. Conclusions The UPLC/Q-TOF-MS technique is a practical and novel method for identifying the fragmentation pattern of metabolites in mango leaf tea by measuring accurate mass with excellent resolution and sensitivity. Funding Sources N/A.

Effect of Moringa oleifera L. Leaf Powder Addition on the Phenolic Bioaccessibility and on In Vitro Starch Digestibility of Durum Wheat Fresh Pasta.

Fresh pasta was formulated by replacing wheat semolina with 0, 5, 10, and 15 g/100 g (w/w) of Moringa oleifera L. leaf powder (MOLP). The samples (i.e., M0, M5, M10, and M15 as a function of the substitution level) were cooked by boiling. The changes in the phenolic bioaccessibility and the in vitro starch digestibility were considered. On the cooked-to-optimum samples, by means of ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry, 152 polyphenols were putatively annotated with the greatest content recorded for M15 pasta, being 2.19 mg/g dry matter (p < 0.05). Multivariate statistics showed that stigmastanol ferulate (VIP score = 1.22) followed by isomeric forms of kaempferol (VIP scores = 1.19) and other phenolic acids (i.e., schottenol/sitosterol ferulate and 24-methylcholestanol ferulate) were the most affected compounds through the in vitro static digestion process. The inclusion of different levels of MOLP in the recipe increased the slowly digestible starch fractions and decreased the rapidly digestible starch fractions and the starch hydrolysis index of the cooked-to-optimum samples. The present results showed that MOLP could be considered a promising ingredient in fresh pasta formulation.

Scopoletin: Antiamyloidogenic, Anticholinesterase, and Neuroprotective Potential of a Natural Compound Present in Argyreia speciosa Roots by In Vitro and In Silico Study.

Alzheimer’s disease (AD) is characterized by depositions of amyloid β (Aβ) peptides aggregates resulting in plaques formation in the central nervous system (CNS). This study evaluates the disease-modifying potential of scopoletin against multiple factors associated with AD such as cholinesterase enzymes, Aβ peptides, and neuroprotective properties against Aβ- and H2O2-induced cytotoxicity under in vitro conditions. Scopoletin was identified and quantified using UPLC-QTOF (ultra-high performance liquid chromatography-quadrupole time-of-flight) and high-performance liquid chromatography (HPLC), respectively. The antiamyloidogenic potential was evaluated by thioflavin T and congo red binding assay. Inhibition of key enzymes, that is, acetylcholinesterase and butyrylcholinesterase, was investigated by Ellman’s assay. UPLC-QTOF analysis showed that most abundant phytoconstituent present in Argyreia speciosa hydroalcoholic root extract was scopoletin followed by festuclavine and ergometrine. Scopoletin was further quantified using novel reverse phase (RP)-HPLC method developed in this study. The neuroprotective potential of scopoletin was found to be 69% against Aβ42-induced neurotoxicity and 73% against H2O2-induced cytotoxicity in PC12 cell culture at 40 μM final concentration. At the same concentration, scopoletin inhibited Aβ42 fibril formation up to 57%. The IC50 concentration for AChE and BuChE enzyme inhibition by scopoletin was 5.34 and 9.11 μM, respectively. The antiaggregation and enzyme inhibition results were complemented with strong molecular interactions of scopoletin with target proteins validated by in silico molecular docking analysis. Based on this study, it can be concluded that scopoletin can be used as a lead for amelioration of symptoms and disease-modifying effects in AD.

Metabolomics-based profiling with chemometric approach to delineate the bio-pharmaceutical properties of fruit extracts from Ligustrum vulgare L

Ligustrum vulgare L. (Family: Oleaceae) is an evergreen shrub native to North-west Africa, Europe and Western Asia. In the present work, infusion (water), ethyl acetate, methanol and n-hexane extracts of the fruits of L. vulgare L. were studied for their in vitro antioxidant and enzyme inhibitory properties along with their untargeted polyphenolic profile (using an ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry approach). Untargeted metabolomics coupled to multivariate statistics showed that Ligustrum fruit extracts were very abundant in polyphenols, such as flavonoids, tyrosols and lignans, with ethyl acetate allowing the highest recovery among the extraction solvents tested. The methanolic extract inhibited the highest acetylcholinesterase (AChE), tyrosinase and glucosidase activity (4.23 mg galatamine equivalent (GALAE)/g, 132.85 mg kojic acid equivalent (KAE)/g and 1.94 mmol acarbose equivalent (ACAE)/g respectively) while the ethyl acetate extract inhibited the highest butyrylcholinesterase (BChE) and amylase activity (4.92 mg GALAE/g and 0.73 mmol ACAE/g, respectively). However, the infusion showed the weakest activity against all the enzymes studied. However, the extract showed highest antioxidant property in the antioxidant assays (except for phosphomolybdenum). The present paper is the first comprehensive report on L. vulgare fruits and thus our findings are of considerable interest to design new bioproducts for the pharmaceutical and cosmeceutical industry in the future.

Fucoidan⁻Fucoxanthin Ameliorated Cardiac Function via IRS1/GRB2/ SOS1, GSK3β/CREB Pathways and Metabolic Pathways in Senescent Mice.

The effects of low molecular weight fucoidan (LMWF) in combination with high-stability fucoxanthin (HSFUCO) on cardiac function and the metabolic pathways of aging mice (Mus musculus) were investigated. We demonstrated that LMWF and HSFUCO could improve cardiac function in aging mice. Aging mice were treated with LMWF and HSFUCO, either on their own or in combination, on 28 consecutive days. Electrocardiography and whole-cell patch-clamp were used to measure QT interval and action potential duration (APD) of the subjects. Cardiac tissue morphology, reactive oxygen species, and Western blot were also applied. Ultra-high-performance liquid chromatography–quadrupole time-of-flight (UPLC-QTOF) mass spectrometry was used for investigating metabolic alterations. The use of LMWF and HSFUCO resulted in improvements in both ventricular rhythms (QT and APD). Treatment with fucoidan and fucoxanthin reduced the expression levels of SOS1 and GRB2 while increasing GSK3β, CREB and IRS1 proteins expression in the aging process. Three main metabolic pathways, namely the TCA cycle, glycolysis, and steroid hormone biosynthesis, were highly enriched in the pathway enrichment analysis. When taken together, the LMWF and HSFUCO treatment improved both the ventricular rhythm and the muscular function of aging subjects by interfering with the metabolism and gene function.

Sorting nexin 10 controls mTOR activation through regulating amino-acid metabolism in colorectal cancer

Amino-acid metabolism plays a vital role in mammalian target of rapamycin (mTOR) signaling, which is the pivot in colorectal cancer (CRC). Upregulated chaperone-mediated autophagy (CMA) activity contributes to the regulation of metabolism in cancer cells. Previously, we found that sorting nexin 10 (SNX10) is a critical regulator in CMA activation. Here we investigated the role of SNX10 in regulating amino-acid metabolism and mTOR signaling pathway activation, as well as the impact on the tumor progression of mouse CRC. Our results showed that SNX10 deficiency promoted colorectal tumorigenesis in male FVB mice and CRC cell proliferation and survival. Metabolic pathway analysis of gas chromatography–mass spectrometry (GC-MS) data revealed unique changes of amino-acid metabolism by SNX10 deficiency. In HCT116 cells, SNX10 knockout resulted in the increase of CMA and mTOR activation, which could be abolished by chloroquine treatment or reversed by SNX10 overexpression. By small RNA interference (siRNA), we found that the activation of mTOR was dependent on lysosomal-associated membrane protein type-2A (LAMP-2A), which is a limiting factor of CMA. Similar results were also found in Caco-2 and SW480 cells. Ultra-high-performance liquid chromatography–quadrupole time of flight (UHPLC-QTOF) and GC-MS-based untargeted metabolomics revealed that 10 amino-acid metabolism in SNX10-deficient cells were significantly upregulated, which could be restored by LAMP-2A siRNA. All of these amino acids were previously reported to be involved in mTOR activation. In conclusion, this work revealed that SNX10 controls mTOR activation through regulating CMA-dependent amino-acid metabolism, which provides potential target and strategy for treating CRC.

Study of grape bioactive stilbenes by suspect screening metabolomics

Stilbenes are one of the main classes of grape polyphenols associated with the beneficial effects of drinking wine. In the present study, a suspect screening metabolomics approach was used to study these grape compounds. Analysis was performed by Ultra High Performance Liquid Chromatography/Quadrupole Time-Of-Flight (UHPLC/QTOF) mass spectrometry and compounds were identified using the database GrapeMetabolomics expressly constructed. By this approach, a total of 18 stilbene derivatives was identified in two V. vinifera red grape varieties (cvs. Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns. Identifications were confirmed by multiple mass spectrometry (MS/MS). Bioactive stilbenes such as trans-resveratrol, Z- and E-piceid, piceatannol and Z- and E-astringin, and several resveratrol dimers, trimers and tetramers such as pallidol and pallidol-3-O-glucoside, E- and Z--viniferin, E- and Z-δ-viniferin, caraphenol B, parthenocissin A, E- and Z-miyabenol C, hopheapenol, ampelopsin H, vaticanol C-like or isohopeaphenol, were identified. In our knowledge, some of these resveratrol trimers and tetramers were reported in grape for the first time.