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Flammulina velutipes is the fourth largest edible fungus in China with high nutritional value. In this paper, ultrahigh-performance liquid chromatography tandem hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-Orbitrap MS) was used to identify the secondary metabolites of F. velutipes. The metabolites were identified by comparing the retention time, accurate molecular weight, and MS2 data with standard databases of mzVault and mzCloud (compound: 17,000+) and BGI high-resolution accurate mass plant metabolome database (plant metabolite: 2500+). Finally, 26 secondary metabolites were preliminarily identified, including flavonoids, phenylpropanoids, organic acids, and steroids.

Kadsura coccinea (Lem.) A.C. Smith has been used as a fruit as well as folk medicine, possessing several effective constituents, including anthocyanins, lignans, and triterpenoids. To the best of our best knowledge, only a few qualitative analyses of anthocyanin from the epicarp of K. coccinea have been reported so far; therefore, it is quite necessary to investigate the kinds of anthocyanins in the epicarp of K. coccinea. In this experiment, plant epicarps were extracted in 0.1% HCl (v/v) in methanol with the help of ultrasonic machine. A rapid, sensitive, and effective method for the identification of constituents was established using UHPLC-Q-Exactive Orbitrap mass spectrometry combined with parallel reaction monitoring (PRM). As a result, a total of 57 anthocyanin derivatives were tentatively characterized from K. coccinea by comparing with standards or studies of retention time, MS data, and MS2 fragments from the literature, fifty-five of these derivatives were reported for the first time. This result developed the constituents database, which will establish a keynote for quality control and pharmacological activity of research in pharmaceutical fields. Moreover, the utilization of the epicarp is beneficial for the environment, reducing the waste of resources.

Background: Fu Zheng Fang (FZF) is an important Chinese medicine prescription for tumor treatment in our hospital, which has two different types, traditional Chinese medicine (TCM) decoction pieces and TCM formula granules. Objective: This study aimed to determine the effective composition of the drug FZF. Methods: In this research, FZF decoction pieces and FZF formula granules were collected and their composition, determined by HPLC-Q-Exactive Orbitrap/MS, and multivariate statistical analysis, was applied to distinguish differential metabolite patterns between two groups. Results: A clear cut difference in the composition of the two groups was observed. 124 differential chemical compositions could be identified in positive mode, while 59 differential chemical compositions could be identified in negative mode. The differential chemical compositions were mainly concentrated in flavonoids, organic acids, fatty acids, amino acids compounds, and presenting different change rules, mainly involved in the flavonoid biosynthesis, flavone and flavonol biosynthesis two metabolic pathways. Conclusion: This study provides basic information that may be of use in the formulation of the drug in different dosages and in the examination of their efficacy.

To systematically analyze the chemical constituents of Citri Sarcodactylis Fructus (CSF) from different origins, an efficient approach based on ultraperformance liquid chromatography plus Q-Exactive Orbitrap tandem mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) detection for the discrimination of chemical components from of 15 batches of CSF from four main origins was used in this research. Through parent peaks, fragment peaks, fragmentation characteristics, and comparative analysis with the literature and reference standards, a total of 77 components from the methanol extracts including 18 coumarins, 24 flavonoids, seven organic acids, three limonoids, and 25 other compounds were detected and identified. Among them, 15 components have not been reported previously in the CSF. Notably, the stachydrine peak initially showed a higher content in the total ion current chromatogram. Overall, CSF produced in the Zhejiang province contained a richer variety of chemical compositions. These observations provided a theoretical basis for the further quality assessment and application of CSF.

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

Potentilla freyniana Bornm. (P. freyniana), belonging to the family Rosaceae, has been used as a folk medicine in China. However, as we know, the constituents and the systematic elucidation of the mechanism were not fully investigated. Therefore, it is necessary to develop a rapid method using LC-MS and network pharmacology for the detection and identification of constituents and the systematic mechanism of P. freyniana. Firstly, the flavonoids were detected and identified based on ultra-high-performance liquid chromatography coupled with Quadrupole-Exactive Focus Orbitrap MS (UHPLC-Q-Exactive Orbitrap MS). After that, the potential targets of those constituents were obtained by database mining. Then, the core targets were predicted by protein-protein interaction network and network analysis. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out via DAVID. This finding revealed that P. freyniana possessed 43 flavonoids (40 of them were first reported) with 23 core target genes, which are associated with PI3K-Akt, MAPK, TNF signaling pathway, and pathway in cancer. This study demonstrated the multicompound, multitarget, and multimechanism of P. freyniana, which are very beneficial to develop the further study and utilization of this plant including the material basis and quality control research.

Lipidomic strategies have been applied to many methodologies, such as species differentiation and lipid-associated disease identification. In this study, we first applied a lipidomic strategy coupled with UPLC-Q-Exactive Orbitrap mass spectrometry to investigate the differences between reconstituted milk and ultra-high temperature sterilization (UHT) milk. Fifteen lipids were selected as biomarkers to identify UHT and reconstituted milk by OPLS-DA method. Furthermore, a discriminant model to identify reconstituted milk from UHT milk was established based on these biomarkers. This robust discriminative model provided a strong technical proof to promote the development of the market norms concerning the identification of UHT and reconstituted milk.

There has been great interest in phospholipids (PLs) from marine by-products due to their long-chain polyunsaturated fatty acids with unique health and functional properties. Here, marine PLs from squid viscera and gonads were comprehensively characterized and compared by UPLC-Q-Exactive Orbitrap/MS-based lipidomics analysis. A total of thirteen phospholipid classes including 1223 molecular species were identified and quantified in both resources. PC, PE and SM were further isolated from the total PLs of squid viscera and gonads, respectively. All isolated squid PL components were first evaluated for anti-inflammatory, antioxidant and cardiovascular effects using in vivo zebrafish models. Our results showed the diversity, content and physiological functions of PLs from squid by-products, which provided a basis for their future application in the nutritional and pharmaceutical industry.

Flos Chrysanthemi Indici (FCI) is a common food and its preparation (FCIP) is generally made to realize the medicinal value of FCI in a more accurate and controllable way. In this paper, an efficient UHPLC Q Exactive HF Hybrid Quadrupole-Orbitrap MS method was exploited to elucidate the difference of chemical components between FCI and FCIP. Multi-fingerprints (HPLC-FP, UV-FP, and DSC-FP) were established with the evaluation results integrated by a weighted mean algorithm using variation coefficient (CVWM), which is more objective and reasonable. Through this method, the quality grades of 25 FCIP samples from four manufacturers were successfully discriminated. Meanwhile, the PLS model for the profile-efficacy relationship between the HPLC-FP and the antioxidant activities was established, as well as the correlation between the antioxidant ability (IC50) and the DSC curve (enthalpy values) were preliminary explored. In conclusion, this study provides a novel and holistic analytical strategy for food herbal remedies and its preparation.

We recently applied untargeted metabolomic profiling on the plasma obtained from consecutive attenders referred for conventional Level 3 home-sleep studies with excessive daytime somnolence, comparing 17 OSAHS patients (AHI≥15, Epworth Score 13.5±4.5) with 16 age, gender, and BMI matched sleepy subjects (sleepy snorers (SS)) with negative home polysomnography tests (AHI We reported 6 biologically plausible plasma metabolites that can differentiate OSAHS from SS of similar phenotype with an AUC of 0.982 (95% CI: 0.9-1.0) (figure 1), with these key metabolites being essential lipids involved in protein synthesis and the formation of antioxidative, antiglycating, and free radical scavenging dipeptides. We now report early changes in these biomarkers following CPAP in those with OSAHS. 11 OSAHS patients with AHI≥15 (63.6% male, Age 54.4±6.9, BMI 34.2±4.0, AHI 47.6±25.6, Epworth 13.7±4.8) were commenced on standard auto-adjustment CPAP devices (Phillips DreamStation set at 4 to 18 cm H2O). Mean use of CPAP was 6.6±1.4 hours and average residual AHI was 6.9±6.0. Plasma was sampled pre and post treatment (42-70 days treatment), and metabolomically assessed using the Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometry platform. 16 sleepy snorers with AHI Our previously reported biomarkers associated with processes such as oxidative stress, inflammation, and dysregulation of energy homeostasis improve with short-term treatment with CPAP towards the level of sleepy snorers of similar age, phenotype, and no OSAHS (figure 2). We feel these metabolites have significant potential in the future care pathways of OSAHS, and could reflect the cardiometabolic risk associated with OSAHS better than current diagnostic modalities. Reference O’Rourke, et al. Plasma metabolomics identifies OSAHS. ERS Congress 2021.

In this work, lipid profile migration from muscle to juice during the tilapia muscle steaming process was revealed by a transactional analysis of data from ultra-high-performance liquid chromatography coupled with Q Exactive (UHPLC-QE) Orbitrap mass spectrometry (MS) and lipidomics. Firstly, the lipids in tilapia muscles and juices at different steaming time points were extracted and examined by UHPLC-QE Orbitrap mass spectrometry. Secondly, a transactional analysis procedure was developed to analyze the data from UHPLC-QE Orbitrap MS and lipidomics. Finally, the corrected lipidomics data and the normalized MS data were used for lipid migration analysis. The results suggested that the transactional analysis procedure was efficient to significantly decrease UHPLC-QE Orbitrap MS workloads and delete the false-positive data (22.4–36.7%) in lipidomics data, which compensated the disadvantages of the current lipidomics method. The lipid changes could be disappearance, full migration into juice, appearance in juice, appearance in muscle, appearance in both muscle and juice, and retention in the muscle. Moreover, the results showed 9 (compared with 52), 5 (compared with 116), and 10 (compared with 178) of lipid class (compared with individual lipid) variables showed significant differences among the different steaming times (0, 10, 30, and 60 min) in all the muscles, juices, and muscle-juice systems, respectively. These results showed significant lipid profile migration from muscle to juice during the tilapia steaming process.

Inula cappa (Buch.-Ham. ex D. Don) DC has been used in traditional Chinese medicine to treat malaria, dysentery, and hepatitis. Previous studies have shown that chlorogenic acid is the effective ingredient of plants in this family. And the research of the chlorogenic acid in Inula cappa will help to further improve the effective resource utilization rate of this plant. Therefore, it is necessary to establish an accurate method to characterize the chlorogenic acid components in Inula cappa. In this study, a simple, fast, and sensitive UHPLC-Q-Exactive Orbitrap mass spectrometry method was established, which can simultaneously analyze known and unknown ingredients in a short time (within 30 minutes) in Inula cappa. According to the diagnosis fragmentation ions, retention time, and bibliography, 68 chlorogenic acid derivatives were identified in Inula cappa. The results of this experiment lay the foundation for the active substances and quality control of Inula cappa and provide a theoretical basis for whether Inula cappa can be an alternative to the endangered wild medicinal materials of the same family.

The root of Reynoutria multiflora (Thunb.) Moldenke (syn: Polygonum multiflorum Thunb.) is a distinguished herb that has been popularly used in traditional Chinese medicine. The raw Reynoutria multiflora (RRM) should be processed by steaming before use, and the processing time is not specified in the processing specification. Our previous studies showed that the efficacy and toxicity of processed Reynoutria multiflora (PRM) at different processing times were inconsistent. A comprehensive identification method was established in this study to find a quality marker of raw Reynoutria multiflora (RRM) and processed Reynoutria multiflora (PRM) with different processing times. Metabolomics based on ultra-high-performance liquid chromatography tandem quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive plus orbitrap MS/MS) was used in this study. Using the CD.2 software processed database, multivariate statistical analysis methods coupled with cluster analysis and heatmap were implemented to distinguish between RRMs and PRMs with different processing times. The results showed that RRM and PRMs processed for 4, 8, 12, and 18 h cluster into group 1, and PRM processed for 24 and 32 h into group 2, indicating that it can effectively distinguish between the two groups and twenty potential markers, made the highest contributions to the observed chemical differences between two groups. Among them, tetrahydroxystilbene-O-hexoside-O-galloyl and sucrose can be used to identify PRM processed for 24 h. Therefore, the properties of RRM changed after 24 h of processing, and the quality markers were screened to distinguish RRM and PPM. It can also be used as an important control technology for the processing of RM, which has wide application prospects.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) has gained popularity in the proteomics field for its capability to improve mass spectrometry sensitivity and to decrease peptide co-fragmentation. The recent implementation of FAIMS on Tribrid Orbitrap instruments enhanced proteome coverage and increased the precision of quantitative measurements. However, the FAIMS interface has not been available on older generation Orbitrap mass spectrometers such as the Q-Exactive. Here, we report the integration of the FAIMS Pro device with embedded electrical and gas connections to a Q-Exactive HF mass spectrometer. Proteomic experiments performed on HeLa tryptic digests with the modified mass spectrometer improved signal to noise and reduced interfering ions, resulting in an increase of 42% in peptide identification. FAIMS was also combined with segmented ion fractionation where 100 m/z windows were obtained in turn to further increase the depth of proteome analysis by reducing the proportion of chimeric MS/MS spectra from 50 to 27%. We also demonstrate the application of FAIMS to improve quantitative measurements when using isobaric peptide labeling. FAIMS experiments performed on a two-proteome model revealed that FAIMS Pro provided a 65% improvement in quantification accuracy compared to conventional LC-MS/MS experiments.

Polygoni Multiflori Radix Praeparata (PMRP), as the processed product of tuberous roots of Polygonum multiflorum Thunb., is one of the most famous traditional Chinese medicines, with a long history. However, in recent years, liver adverse reactions linked to PMRP have been frequently reported. Our work attempted to investigate the chemical constituents of PMRP for clinical research and safe medication. In this study, an effective and rapid method was established to separate and characterize the constituents in PMRP by combining ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Based on the accurate mass measurements for molecular and characteristic fragment ions, a total of 103 compounds, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other compounds were identified or tentatively characterized. Forty-eight compounds were tentatively characterized from PMRP for the first time, and their fragmentation behaviors were summarized. There were 101 components in PMRP ethanol extract (PMRPE) and 91 components in PMRP water extract (PMRPW). Simultaneously, the peak areas of several potential xenobiotic components were compared in the detection, which showed that PMRPE has a higher content of anthraquinones and stilbenes. The obtained results can be used in pharmacological and toxicological research and provided useful information for further in vitro and in vivo studies.

The current study aimed to explore the mechanism of Guizhi Shaoyao Zhimu Decoction in the treatment of gouty arthritis based on the combination of Vanquish Ultra High Performance Liquid Chromatography Q Exactive IV Pole-electrostatic field orbital trap high resolution mass spectrometer (UHPLC-Q-Orbitrap HRMS) and network pharmacology. Firstly, UHPLC-Q-Orbitrap HRMS was established and applied to separation and identification of the chemical components of GSZD. Then, using network pharmacology analysis to obtain the potential active components and candidate targets underlying the effect of GSZD on gouty arthritis (GA). Through targeted and untargeted analysis, a total of 79 components, including 3 chromones, 26 flavonoids, 10 alkaloids, 10 phenolic acids, 7 nucleosides and nucleobases, and 23 other components, were characterized from GSZD. 11 compounds were considered to potentially key active compounds against GA which may be via interacting with multiple inflammatory factors and multiple inflammatory pathways.

Introduction Erycibe obtusifolia and E. schmidtii are widely used in traditional Chinese medicine (TCM) to treat joint pain and rheumatoid arthritis. With the reduction of wild E. obtusifolia and E. schmidtii resources, Porana sinensis has been widely used as a substitute. However, few studies have been conducted on the chemical composition and quality control of P. sinensis. Objective To clarify the chemical composition and improve the quality control of P. sinensis. Methodology We developed an ultra-high performance liquid chromatography electrospray ionisation Q-Exactive Focus tandem mass spectrometry (UHPLC-ESI-Q-Exactive Focus-MS/MS) method to characterise the chemical constituents of P. sinensis. A strategy based on a combination of high-performance thin-layer chromatography (HPTLC) and direct analysis in real-time (DART) ion source was proposed for the identification of alkaloid components in P. sinensis. Thin-layer chromatography (TLC) autography for 2,2'-diphenyl-1-picrylhydrazyl free radical (DPPH˙) and TLC bioautography for xanthine oxidase were used to rapidly screen marker compounds for high-performance liquid chromatography (HPLC) determination of P. sinensis. Based on the selected marker compounds, a HPLC method for the quantitative determination of eight marker compounds in P. sinensis was developed. Results Eighteen compounds in P. sinensis were identified by UHPLC-Q-Exactive MS. Taken together with the results of TLC autography and TLC bioautography, eight compounds were chosen as marker compounds for HPLC determination of P. sinensis. The alkaloid components in P. sinensis were identified as Baogongteng A and Baogongteng C by DART-MS. Conclusion We systematically clarified the chemical composition of P. sinensis for the first time, and potentially improved its quality control. These results should promote the application of P. sinensis as a new resource for Caulis Erycibes.

Background Microbial fermentation has been widely applied in traditional Chinese medicine (TCM) for thousands of years in China. Various beneficial effects of fermentation for applications in TCM or herbals have been reported, such as enhanced anti-ovarian cancer, antioxidative activity, and neuroprotective effects. Ge-Gen-Qin-Lian decoction (GQD), a classic TCM formula, has been used to treat type 2 diabetes mellitus in China. In this study, GQD was fermented with Saccharomyces cerevisiae, and the antidiabetic activities and overall chemical profiles of raw and fermented GQD (FGQD) were systematically compared. Methods First, the antidiabetic effects of GQD and FGQD on high-fat diet and streptozotocin (STZ)-induced diabetic rats were compared. Then, high-performance liquid chromatography Q Exactive MS was applied for rapid characterization of the chemical components of GQD. Additionally, we proposed an integrated chromatographic technique based untargeted metabolomics identifying differential chemical markers between GQD and FGQD and targeted analysis determining the fermenting-induced quantitative variation tendencies of chemical marker strategy for overall chemical profiling of raw and fermented GQD. Results Both GQD and FGQD displayed effects against HFD and STZ-induced diabetes, and FGQD showed a better recovery trend associated with profound changes in the serum lipoprotein profile and body weight gain. In addition, 133 compounds were characterized from GQD. It was demonstrated that the integrated strategy holistically illuminated 30 chemical markers contributed to the separation of GQD and FGQD, and further elucidated the fermenting-induced chemical transformation mechanisms and inherent chemical connections of secondary metabolites. Although there were no new secondary metabolites in FGQD compared with GQD, the amounts of secondary metabolites, which were mostly deglycosylated, were redistributed in FGQD. Conclusion The anti-diabetic activities of GQD could be improved by applying fermentation technology. Moreover, the proposed strategy could serve as a powerful tool for systematically exploring the chemical profiles of raw and fermented formulas. Electronic supplementary material The online version of this article (10.1186/s13020-018-0208-7) contains supplementary material, which is available to authorized users.

Marine phospholipids (MPLs) are valuable components that can be applied within diverse areas like nutrition, pharmacy, and medicine as well as basic scientific research, particularly due to their high concentration of long chain polyunsaturated fatty acids. In this work, lipidomics approach by UPLC-Q-Exactive Orbitrap/MS was used for the identification, quantification, comparison, and characterization of phospholipids (PLs) from three different marine resources (shrimp head, codfish roe, and squid gonad). In total, 310 PL molecular species containing 34 different structures of fatty acid chains were identified simultaneously by Lipidsearch software. Significant differences between three groups in the PL classes, compositions and contents were revealed. A list of characteristic PL species that represent significant differences among groups was determined by lipidomics analysis. Until now, the information about the deep comprehensive description of PL from marine sources is limited. Thus, this study will give a very potential starting point to develop MPL products and establish the quality standards for different marine raw materials.

Gibberellic acid (GA3) is widely applied in agriculture and food worldwide. Profiling the degradation products and their formation pattern under stress are helpful for deeply understanding GA3 regulating plant physiology and GA3 safety in agricultural crops. This study firstly investigated the degradation behavior of GA3. Different stress factors such as light, pH and temperatures were investigated through photolysis and hydrolysis experiments. Five degradation products were identified using ultra high-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry (UHPLC Q-Exactive Orbitrap MS). Three degradation products were produced under ultraviolet photolysis conditions. Two isomers (iso-GA3 and gibberellenic acid) were formed under alkaline conditions. In order to characterize each degradation product, complete mass fragmentation pathways of all analytes were initially established. These results could provide a practical reference for the safety of agricultural products and the guidance for scientific application of GA3 and proposed storage conditions of GA3.

Among natural freshwater pollutants, cyanotoxins, mycotoxins, and phytotoxins are the most important and less studied. Their identification in surface water is challenging especially cause of the lack of standards and established analytical parameters. Most target methods focus one or a single group of compounds with similar characteristics. Here we present an AIF fast method for the tentative identification of natural toxins in water. Respect to the previous method [1], it offers higher performances for the acquisition of unknown compounds at low levels for higher number of analytes. The key aspects of the method are: • The qualitative screening DIA-AIF workflow using Q Exactive Orbitrap. Both targeted and suspect screening bases have been combined with online databases and suspect list to retrieve candidates as suspect natural toxins and their metabolites or degradation products. • The in-silico analysis of mass spectrums allowed a fast structural characterization. • The workflow has been finally applied to real samples coming from the Czech Republic, Italy, and Spain allowing the determination of 17 suspect natural toxins, 4 of them confirmed. None toxin passed the limit of 1 µg/L taken from the legislation applied for microcystin LR and arbitrarily extended to all toxins., This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 722493 (NaToxAq); and by the Generalitat de Catalunya (Consolidated Research Group “2017 SGR 1404 - Water and Soil Quality Unit”). We also want to thank the Recetox colleagues Petra Laboha, Eliska Sychrova and Barbara Kubíčková for the sampling, logistics and supply of the water samples from Czech Republic.

In this study, a rapid and reliable method based on ultrahigh-performance liquid chromatography coupled with Q Exactive HF-X mass spectrometry (UHPLC-QE/MS) was established for the simultaneous quantification and validation of acrylamide, 5-hydroxymethylfurfural, and 14 heterocyclic aromatic amines in thermally processed foods. With the optimization of the pretreatment method, all 16 hazardous compounds with different polarities were simultaneously extracted and purified by one-step purification. By studying various acquisition modes in detail, full MS + PRM detection using an electrospray ionization source in the positive mode gives an excellent-shaped chromatographic peak and thereby achieves a better quantitative ability for analytes in the matrix. This method demonstrated good quantification recovery in the range of 68.85-146.42%. The limits of quantification were within the range from 0.1 to 50 ng/mL. With the method proposed, the simultaneous determination of 16 hazardous compounds in different thermally processed foods was successfully applied. The all-fragment-ion approaches at high resolution have the ability to reduce false-positive peak detections arising from peak alignment software in the detection of samples significantly. The proposed isotope dilution UHPLC-QE/MS method was validated and demonstrated to be sensitive, accurate, and precise for the simultaneous quantification of multiple contaminants in one injection.

Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."

Artemisia argyi Lévi. et Vant is a traditional Chinese medicine with a long history, and its buds and seedlings can be used as vegetables. However, the investigations on the chemical constituents of A. argyi are not sufficient. In this paper, ultra-high performance liquid chromatography tandem hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive-MS/MS) was used to identify the chemical constituents of A. argyi. The Q Exactive mass spectrometer was used to collect MS and MS2 data. Finally, 125 compounds were preliminarily identified in A. argyi by comparing the retention time and accurate molecular weight with standard databases such as MZVault, MZCloud, and BGI Library (self-built standard Library by BGI Co., Ltd), including flavonoids, phenylpropanoids, terpenoids, and organic acids.

Aurantio-obtusin (AO) is a major anthraquinone compound isolated from Cassiae Semen or Duhaldea nervosa, which possesses diverse pharmacological effects. Previous studies have shown that it has a good effect on lowering blood lipids and treating various diseases. A few studies have also reported about its metabolites. A rapid and reliable method using ultra-high-performance liquid chromatography-Q-Exactive Orbitrap mass spectrometry and multiple data-processing technologies was established to investigate the metabolites of AO in the plasma and various tissues of rats, including the heart, liver, spleen, lung, kidneys, and brain. Finally, a total of 36 metabolites were identified in the plasma of rats, which could be very beneficial for understanding the effective form of AO metabolites leading to new drug discovery. The result demonstrated that this strategy, especially parallel reaction monitoring, has shown a wide range of applications in the identification of metabolites.

Ligustri Lucidi Fructus (LLF) is the dried and mature fruit of Ligubtrum lucidum Ait., which has the effect of nourishing the liver and kidney, brightening the eyes and promoting the growth of black hair. Wine-processed LLF is commonly used in traditional Chinese medicine; however, the processing mechanisms are still unclear. Herein, a system data acquisition and mining strategy was designed to investigate the chemical profile differences between the raw and wine-processed LLF, based on high-performance liquid chromatography-Orbitrap high resolution mass spectrometry coupled with multivariate statistical analysis including principal component analysis and partial least square analysis. Afterwars, a total of 55 components were found to be the main contributors to the significant difference between raw and wine-processed LLF by comparison with chromatographic behaviors, intact precursor ions, and characteristic MS fragmentation patterns. In addition, 10 main constituents of raw and wine-processed LLF were simultaneously determined by UHPLC-MS/MS for analyzing the content variations. Some structural transformation mechanisms during wine processing were deduced from the results. The results may provide a scientific foundation for deeply elucidating the wine-processing mechanism of LLF.

Sphingolipids (SLs) are endogenously bioactive molecules with diverse structures, and its metabolic disorders are involved in the progression of many diseases. In this study, an ultra-performance liquid chromatography quadrupole exactive mass spectrometry (UPLC-Q-Exactive-MS) method was established to comprehensively profile SLs in plasma. First, the fragment patterns of SL standards of each subclass were investigated. Then, the SL species in plasma were characterized based on the fragmentation rules. Finally, a total of 144 endogenous SL species consisting of 216 regioisomers were identified in plasma of human, golden hamster and C57BL/6 mice, which was the most comprehensive identification for SLs in plasma. In addition to the known species, 19 SL species that have never been reported were also identified. The profile of SLs in plasma of human and two rodent species was compared subsequently. It was worth noting that a total of 9 SL molecular species consisting of 11 regioisomers with low abundance were successfully identified in human plasma through comparison among species. Those findings contribute to a deeper understanding of SLs in human plasma and provide scientific basis for the selection of animal model. The established profile of SLs in plasma could be used for screening of lipid biomarkers of various diseases.

The lipophilic compounds product (LCP), which was isolated and purified from Eucalyptus viminalis plants, has shown earlier broad antimicrobial and anti-inflammatory activities. To study secondary compounds responsible for the pharmacological activities, chemical composition of the LCP was studied with application of ultra-performance liquid chromatography combined with photodiode array detector and high-resolution Q Exactive Orbitrap mass spectrometer (UPLC-PDA-HRMS/MS). There were found thirty two compounds: twenty phloroglucinol derivatives (isopentyl diformyl phloroglucinol, macrocarpals, sideroxylonals, etc.), eight ursane type triterpenoids (loxanic acid, dehydroursolic acid lactone, dehydroursolic acid lactone acetate, two isomers of p-coumaroyl-dehydroursolic acid lactone and two isomers of feruloyl-dehydroursolic acid lactone), sequiterpenoid (S)-β-macrocarpene and three unknown phenolics. The major compounds of the LCP were pharmacologically active macrocarpals A and B, dehydroursolic acid lactone and its derivatives. It is supposed that previously discovered antimicrobial and anti-inflammatory activities of the LCP is due to the high contents of these secondary compounds., Eucalyptus viminalis; Lipophilic compounds product; Phloroglucinol derivatives; Triterpenoids; UPLC-PDA-HRMS/MS.

BackgroundAlthough the clinical effect of stir-fried Dolichos lablab L. kernel has been approved in modern traditional Chinese medicine, existing associated studies mainly focus on its clinical studies and chemical ingredients. However, there are few studies on pharmacodynamics material basis and molecular mechanism of stirfried Dolichos lablab L. kernel in treatment of type-2 diabetes(T2DM), thus restricting the further development and utilization of stir-fried Dolichos lablab L. kernel.MethodsA qualitative analysis on saponin chemical ingredients of stir-fried Dolichos lablab L. kernel was performed using UHPLC-Q-Exactive Orbitrap MS. A total of 10 saponin ingredients were selected. Moreover, target screening, biological process and metabolism pathway analysis were accomplished by network pharmacology. Four key proteins(EGFR, IGF1, MAPK1 and PIK3R1) of type-2 diabetes were selected for molecular docking verification with saponin ingredients. Specifically, molecular dynamics simulation of ingredients which have strong bindings with proteins was conducted. ResultsIn this study, 16 saponin ingredients were identified from stir-fried Dolichos lablab L. kernel. There were 91 intersection targets and the KEGG pathway enrichment involved 20 relevant pathways. According to the molecular docking verification, saponin ingredients of stir-fried Dolichos lablab L. kernel can form stable binding with key protein targets. The molecular dynamics simulation further verifies stability and reasonability of the docking results. ConclusionsThis study provides references to identification of efficient ingredients of stir-fried Dolichos lablab L. kernel, screening of quality markers and explanation of relevant action mechanism by combining UHPLC-Q-Exactive Orbitrap MS and network pharmacology.

Content Isochlorogenic acid A, one of the main components of Duhaldea nervosa (Wallich ex Candolle) A. Anderberg (Asteraceae), is a folk medicine used to treat a variety of diseases including fracture and rheumatoid arthritis. Despite its widespread use, the metabolism of isochlorogenic acid A in vivo has not been fully studied. Objective An analytical strategy based on UHPLC-Q-Exactive Orbitrap MS is proposed for the detection and identification of the metabolites of isochlorogenic acid A in rats. Materials and methods Six male Sprague-Dawley rats (180 ± 20 g) were randomly divided into two groups. Then, blood and tissue samples were obtained after oral administration of isochlorogenic acid A (200 mg/kg). All the samples were pre-treated by the Solid Phase Extraction (SPE) method. Next, the samples were analysed by UHPLC-Q-Exactive Orbitrap MS. Finally, the metabolites were identified based on the metabolomic workflow template. Results A total of 33 metabolites were identified in rat plasma, with 30 of them being reported for the first time. The distribution of all metabolites in tissues was first investigated, three of them were widely distributed in liver, lungs, and kidneys. The corresponding reactions including methylation, hydrolysis, sulphate conjugation, glucuronide conjugation, as well as their composite reactions, are reported in this study. Discussion and conclusions This method has wide-scale application prospects in the identification of metabolites. Considering that limited research has been conducted in this area, this study proposes metabolic pathways to further understand mechanisms of isochlorogenic acid A and the forms that are truly effective in vivo.

Rationale The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. Methods Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. Results Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. Conclusions The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

Background: Sargentodoxa cuneata is an herb commonly used by the Hmong people, and many preparations containing this herb have been marketed. However, the source of medicinal materials is not standardized during the production process, and the quality of medicinal materials from different sources may be inconsistent. This study was aimed to establish a qualitative and quantitative analysis method for quality assessment of Sargentodoxa cuneata in different regions. Methods: HPLC/Q Exactive Plus MS was performed in positive mode and negative mode used for qualitative analysis, and there were twenty-one ingredients were tested. Nine high-content or characteristic ingredients were screened out and applied to the quantitative analysis of thirty batches of medicinal materials from different origins. Based on the results of the quantitative analysis, further multivariate statistical analyses were carried out for quality assessment of Sargentodoxa cuneata . Results: Results showed that there were significant differences in the contents of the nine ingredients of Sargentodoxa cuneata in the eastern and southwestern regions. The content of hydroxytyrosol, salidroside, chlorogenic acid, desrhamnosyl isoacteoside, and liriodendrin of Sargentodoxa cuneata in eastern region was higher, while the content of protocatechin in southwestern region was higher. The different ingredients included protocatechuic acid, desrhamnosyl isoacteoside, liriodendrin, and hydroxytyrosol. These four ingredients were important factors that caused the difference in the quality of Sargentodoxa cuneata from different origins. Conclusion: Protocatechuic acid, desrhamnosyl isoacteoside, liriodendrin, and hydroxytyrosol may become a new marker for quality assessment of Sargentodoxa cuneata.

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

The root of Gentiana straminea Maxim. (Gentianaceae), is officially listed as &ldquo, Qin-Jiao&rdquo, in the Chinese Pharmacopoeia for the treatment of rheumatic arthritis, icteric hepatitis, constipation, pain, and hypertension. To establish the geographical origin traceability in G. straminea, its chemical profiles were determined by a UPLC-Q exactive mass spectrometer, from which 43 compounds were identified by comparing retention times and mass spectrometry. Meanwhile, a pair of isomers (loganin and secologanol) was identified by mass spectrometry based on their fragmentation pathway. A total of 42 samples from difference habitats were determined by an UPLC-Q exactive mass spectrometer and the data were assayed with multivariate statistical analysis. Eight characteristic compounds were identified to determine the geographical origin of the herb. To estimate the key characteristic markers associated with pharmacological function, the inhibiting activities of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages were examined. This finding is crucial in realizing the determination of botanical origin and evaluating the quality of G. straminea.

Duhaldea nervosa (D. nervosa) has been used for treatment of bone fracture by external use. Thus, the percutaneous absorption was crucial to the effect of D. nervosa, especially the constituents of percutaneous absorption. However, the constituents in vivo were never investigated to date. In this study, an efficient method was developed for the identification of constituents of percutaneous absorption using UHPLC-Q-Exactive Orbitrap MS and microdialysis technique. A total of 20 constituents including 15 chlorogenic acid analogues, 3 amino acids, and 2 organic acids were unambiguously or tentatively identified based on high-resolution mass data including MS and MS2, chromatography retention time, and bibliography data. To the best of our knowledge, this is the first study to report the constituents of percutaneous absorption from D. nervosa, which will be very helpful for understanding the bioactive compounds and quality control.

Summary: Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules. We found the tyrosine phosphatase Shp-2 to be the master regulator of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates, including Rasa1 and Cortactin with increased pTyr and Gab1 and Erk1/2 with decreased pTyr. Our study demonstrates that large-scale quantitative phosphoproteomics can precisely dissect tightly regulated kinase-phosphatase signaling networks. : Batth et al. use mass spectrometry-based phosphoproteomics to analyze receptor tyrosine kinase signaling activated by different ligands, identifying hundreds of differentially regulated phosphotyrosine sites. Tyrosine phosphatase Shp-2 regulates global tyrosine phosphorylation in a Pdgf-receptor-dependent manner, affecting cellular outcomes. Keywords: phosphoproteomics, Shp-2, PDGF, SHP099, Q exactive, orbitrap, label-free quantitation, tyrosine phosphorylation, TiO2, mass spectrometry