High-resolution Liquid Chromatography-tandem Mass Spectrometry Research Articles

Profiling the alterations of serum proteome in dairy cows with retained placenta using high-throughput tandem mass tags quantitative approach

Background Retained placenta (RP), a quite common disorder in dairy cows, shows a high negative impact on their health status and milk production. Aim To investigate the difference in the serum proteome between the cows with RP and the physiologic puerperium (PP). Material & Methods Analysis of serum samples from nine cows with RP and six with PP using high-resolution liquid chromatography-tandem mass spectrometry approach. The proteins differing in the relative abundance between the PP and RP groups were classified using the Protein Analysis Through Evolutionary Relationship tool. For the pathway enrichment analysis, the REACTOME tool, with the human genome as the background, was employed. The criterion for significance was the false discovery rate corrected P-value less than 0.05. Results In total 651 proteins were identified with altered relative abundance of ten proteins. Among them, seven had higher, and three showed lower relative abundance in RP than in the PP group. The differently abundant proteins participated in 15 pathways: six related to hemostasis, three involved in lipoprotein metabolism, and the remaining ones associated with for instance redox homeostasis, post-translational modification, and scavenging. Finally, the validation of the proteomic results showed that haptoglobin and lipopolysaccharide-binding protein levels reliably differentiated between the RP and PP groups. Conclusion The pattern of serum proteome alterations in the cows with RP mirrored several interplaying mechanisms underlying the systematic response to the presence of RP, therefore representing a source to mine for predictive or prognostic biomarkers.

ヒト肝ミクロソームによる新規デザイナードラッグ1cP-LSDの代謝

A newly encountered designer drug “N1-cyclopropylmethanoyl-lysergicaciddiethylamide (1cP-LSD)” is an acylated derivative of LSD. Based on the structural characteristics, 1cP-LSD is expected to be readily metabolized to LSD, which can make it difficult to discriminate between intakes of 1cP-LSD and LSD. In this study, to obtain the fundamental information needed for proving intake of 1cP-LSD, the metabolism experiments were performed using human liver microsomes (HLM). 1cP-LSD and its 10 metabolites were detected in HLM reaction mixtures of 1cP-LSD using liquid chromatography-high resolution-tandem mass spectrometry with a C18 semi-micro column and the time-course changes in concentrations of them were observed during the first 120 minutes of the reactions. The results demonstrated that 1cP-LSD rapidly decreased in microsomal mixtures (half-life time: 5 min) and a metabolic reaction by carboxylesterase, expressed abundantly in liver, was mostly responsible for the decrease. This short half-life of 1cP-LSD implied biological samples such as urine and blood should be collected from suspects as soon as possible after intake for detecting unchanged 1cP-LSD, which can lead to the proof of intake. Four out of 10 microsomal metabolites (hydroxy-, N-desethyl-, N6-desmethyl and N-desethyl-N6-desmethyl metabolites) could also be useful indicators for proving intake of 1cP-LSD since they retain a cyclopropylmethanoyl group. Especially, the N6-desmethyl metabolite appeared to be a more appropriate indicator because it could be detected in microsomal mixtures over longer periods.

Determination of porcine derived components in gelatin and gelatin-containing foods by high performance liquid chromatography-tandem mass spectrometry

Detection and quantitation of porcine derived components in bovine and donkey gelatins and gelatin-containing foods is required for religious practices and consumer economic interests. In this study, shot-gun proteomics approaches were implemented to identify species-specific peptide biomarkers of three main gelatin origins, including porcine, bovine and donkey. Tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography-tandem high-resolution mass spectrometry (UPLC-HRMS), and 11 peptide biomarkers were selected for porcine, bovine and donkey. The gelatin pretreatment methods in three different food matrixes were optimized, and a label-free quantitative method using the MRM method of the most sensitive biomarker peptides was established. Among them, two porcine gelatin quantitative peptides were confirmed to have good linearity. The method has been successfully applied to the adulteration detection of porcine-derived components in gelatin and the quantification of porcine gelatin in candy and dairy products, with LODs of 0.07, 0.08 and 0.12 mg/kg, respectively. • A strategy for identification of peptide biomarkers of common gelatin species was established and improved with untargeted and targeted proteomics approaches. • Eleven species-specific peptide biomarkers were selected for porcine, bovine and donkey gelatin. • The pretreatment method of gelatin in different food matrixes has been optimized, especially for gelatin in dairy products. • The label-free MRM method is used for quantitative determination of adulterated porcine gelatin in mixed gelatin and gelatin-containing food, with good linearity, specificity, sensitivity and repeatability.

Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

Biomarkers of 4-hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) intake identified from human hepatocyte incubations

ABSTRACT Background 4-Hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) is a psychedelic tryptamine whose use is regulated in several countries. Due to unspecific effects, consumption can be ascertained only through toxicological analyses. However, the trace amounts of tryptamines are usually challenging to detect in biological samples. 4-OH-MPT metabolism was characterized to identify optimal metabolite markers of intake in clinical/forensic toxicology. Research design and methods 4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulate in vivo conditions; samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and data were processed with Compound Discoverer from Thermo Scientific. LC-HRMS/MS and data mining were supported by in silico metabolite predictions (GLORYx). Results Three phase I and four phase II metabolites were identified, including N-oxidation and N-demethylation at the alkylamine chain, and O-glucuronidation and sulfation at the hydroxylindole core. Conclusions 4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT-N-oxide and 4-hydroxy-N,N-propyltryptamine (4-OH-PT) as metabolite biomarkers of 4-OH-MPT consumption after glucuronide/sulfate hydrolysis in biological samples to improve detection of 4-OH-MPT and phase I metabolites; 4-OH-MPT-glucuronide is suggested as an additional biomarker when hydrolysis is not performed. Further research on the metabolism of structural analogues is necessary to evaluate the specificity of 4-OH-MPT metabolite biomarkers.

An analytical platform for the screening and identification of pyrrolizidine alkaloids in food matrices with high risk of contamination

An analytical platform for the detection of pyrrolizidine alkaloids (PAs) in honey, pollen, teas, herbal infusions, and dietary supplements is proposed; it includes a wide-scope suspect screening method, based on a diagnostic product ion filtering strategy for the characterization of PAs, and a target screening and identification method for the high-throughput detection of 118 PAs of a high-resolution mass spectral library. Salting-out assisted liquid–liquid extraction of aqueous extracts combined to ultra-high performance liquid chromatography–high-resolution tandem mass spectrometry was employed. The limit of identification (0.6–30 µg kg−1) of 28 standards were fit-for-purpose in PA-monitoring applications, with a false negative rate <1.3 % at 4 µg L–1. The wide-scope suspect screening method allowed the tentative identification of 88 compounds. The screening of 282 commercial samples revealed a broad contamination of the studied matrices, demonstrating the effectiveness of the platform in detecting and identifying both target and untarget PAs.

Novel Method for Comprehensive Annotation of Plant Glycosides Based on Untargeted LC-HRMS/MS Metabolomics.

Glycosides are a large family of secondary metabolites in plants, which play a critical role in plant growth and development. Due to the complexity and diversity in structures and the limited availability of authentic standards, comprehensive annotation of the glycosides remains a great challenge. In this study, using maize as an example, a deep annotation method of glycosides was proposed based on untargeted liquid chromatography-high-resolution tandem mass spectrometry metabolomics analysis. First, knowledge-based in silico aglycone and glycosyl/acyl-glycosyl libraries were built. A total of 1240 known and potential aglycones from databases and literature were recorded. Next, the MS parameters beneficial to aglycone ion-rich MS/MS were explored using 1782 high-resolution MS/MS spectra of glycosides from the MassBank of North America (MoNA) and confirmed by 52 authentic glycoside standards. Then, screening rules for aglycon ions in MS/MS were recommended. Glycoside candidates were further filtered by MS/MS-based chemical classification and MS/MS similarity of aglycon-glycoside pairs. Finally, the glycosylation sites of flavonoid mono-O-glycosides were recommended by characteristic fragmentation patterns. The developed method was validated using glycosides and nonglycosides from the MoNA library. The annotation accuracy rates were 96.8, 94.9, and 98.0% in negative ion mode (ESI-), positive ion mode (ESI+), and the combined ESI- & ESI+, respectively. The annotation specificity was 99.6% (ESI-), 99.6% (ESI+), and 99.2% (ESI- & ESI+). A total of 274 glycosides (including 34 acyl-glycosides) were tentatively annotated in maize by the developed method. The method enables effective and reliable annotation for plant glycosides.

Bacteroides fragilis derived metabolites, identified by molecular networking, decrease Salmonella virulence in mice model.

In the gut microbiota, resident bacteria prevent pathogens infection by producing specific metabolites. Among bacteria belonging to phylum Bacteroidota, we have previously shown that Bacteroides fragilis or its cell-free supernatant inhibited in vitro Salmonella Heidelberg translocation. In the present study, we have analyzed this supernatant to identify bioactive molecules after extraction and subsequent fractionation using a semi-preparative reversed-phase Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS). The results indicated that only two fractions (F3 and F4) strongly inhibited S. Heidelberg translocation in a model mimicking the intestinal epithelium. The efficiency of the bioactive fractions was evaluated in BALB/c mice, and the results showed a decrease of S. Heidelberg in Peyer's patches and spleen, associated with a decrease in inflammatory cytokines and neutrophils infiltration. The reduction of the genus Alistipes in mice receiving the fractions could be related to the anti-inflammatory effects of bioactive fractions. Furthermore, these bioactive fractions did not alter the gut microbiota diversity in mice. To further characterize the compounds present in these bioactive fractions, Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS) data were analyzed through molecular networking, highlighting cholic acid (CA) and deoxycholic acid. In vitro, CA had inhibitory activity against the translocation of S. Heidelberg by significantly decreasing the expression of Salmonella virulence genes such as sipA. The bioactive fractions also significantly downregulated the flagellar gene fliC, suggesting the involvement of other active molecules. This study showed the interest to characterize better the metabolites produced by B. fragilis to make them means of fighting pathogenic bacteria by targeting their virulence factor without modifying the gut microbiota.

Untargeted analysis of environmental contaminants in surface snow samples of Svalbard Islands by liquid chromatography-high resolution mass spectrometry

In recent years, there is increasing attention on the contaminants of emerging concern (CECs), which include plasticizers, flame retardants, industrial chemicals, pharmaceuticals, and personal care products, since they have been detected even far away from pollution sources. The polar regions are not exempt from the presence of anthropogenic contaminants, and they are employed as a model for understanding the pollutant fate and impact.During the 2021 spring campaign, sixteen surface snow samples were collected close to the research station of Ny-Ålesund located on the Spitsbergen Island of the Norwegian Svalbard Archipelago. The samples were extracted by solid-phase extraction and analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) following an untargeted approach. Compound tentative identification was obtained with the aid of the software Compound Discoverer, using both mass spectral database search and manual validation. Among the 114 compounds identified with a high confidence level in the snow samples, >80 have some commercial or industrial use (drugs, plasticizers, fragrances, etc.), therefore they could be of anthropogenic origin. Nonetheless, a clear contamination trend did not appear in the snow samples collected on eight different days during one month. The comparison with aerosol samples collected in the same area did not help identifying the source, either, since only a few compounds were in common, and they were mainly of natural origin. As such, the analysis of aerosol sample did not support possible long-range transport, also considering that compounds were detected mostly in the coarse fraction.

A novel method for efficient screening and annotation of important pathway-associated metabolites based on the modified metabolome and probe molecules

植物次生代谢物在抵御生物/非生物胁迫、生物间互作以及信息传递等方面发挥重要作用,次生代谢途径解析对植物分子育种、天然产物合成等方面具有重要意义。液相色谱-高分辨串联质谱(LC-HRMS/MS)为次生代谢物鉴定及途径表征提供了技术手段。非靶向LC-HRMS/MS方法可获得丰富的质谱信号,包括一级质谱和二级质谱(MS, MS/MS),但受质谱数据库规模以及次生代谢物复杂性的制约,次生代谢物注释十分困难。该研究以玉米叶片中苯丙烷途径代谢物为例,发展用于非靶向代谢组数据中重要途径代谢物的高效筛选和注释新方法。首先,利用公共代谢途径数据库及文献获取参与苯丙烷代谢途径的61种修饰反应类型,进而从非靶向实验数据中筛选出修饰代谢组。其次,获取开源串联质谱数据中的苯丙烷类化合物作为探针分子,构建探针分子质谱数据库。将探针分子与修饰代谢组共建分子网络,锁定目标途径代谢物并注释结构。该方法在正、负离子模式下分别筛选出玉米叶片中392个和417个苯丙烷途径候选代谢物,去冗余后共注释出129个代谢物,涉及苯丙烷代谢的主要分支途径,如黄酮途径的8个类黄酮、19个氧苷类黄酮和32个碳苷类黄酮,31个羟基肉桂酸途径代谢物以及22个木脂素途径代谢物;其中26个在PubChem和SciFinder数据库中未见收录。该研究利用探针分子结合修饰组可快速锁定途径代谢物,且有助于快速、准确的网络传播注释,可显著提高目标途径代谢物筛选与注释效率,为植物次生代谢途径的深入解析提供分析手段。

β'-Phenylfentanyl Metabolism in Primary Human Hepatocyte Incubations: Identification of Potential Biomarkers of Exposure in Clinical and Forensic Toxicology.

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β'-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β'-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate β'-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β'-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β'-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest β'-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β'-phenylfentanyl use. In silico predictions were mostly wrong, and β'-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β'-phenylfentanyl-positive cases to provide authentic samples.

4-Hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) in vitro human metabolism

Tryptamines are indolealkylamines sharing their core structure with serotonin. Most tryptamines are psychoactive hallucinogens. Every year, new molecules are synthesized to circumvent the laws. To date, 53 tryptamine analogues are monitored by the EU Early Warning System, and, although the total number of intoxications and fatalities are still low, they are increasing. Tryptamines are generally potent and their concentrations in biological matrices are low, making detection challenging in analytical toxicology. Urinary metabolite biomarkers can be targeted to improve detection. However, like other novel psychoactive substances (NPS), there are no data available on the metabolism of the most recently encountered molecules (Malaca. International Journal of Molecular Sciences 2020;21:9279). 4-hydroxy-N,N-methylpropyltryptamine (4-OH-MPT), also known as meprocin, is a psychedelic tryptamine first identified in seizures in Europe in 2018. 4-OH-MPT intake was never reported in the literature or early warning systems in authentic cases, possibly due to lack of specific metabolite biomarkers. The aim of this research was to characterize 4-OH-MPT metabolism in human hepatocytes and identify optimal metabolite markers to identify 4-OH-MPT intake. Analyses were conducted following our in-house protocol (Di Trana. Talanta 2021;235:122740). To assist in metabolite identification, 4-OH-MPT metabolic fate was predicted using GLORYx freeware, generating a list of potential first- and second-generation metabolites with a probability score. 4-OH-MPT was incubated with cryopreserved ten-donor-pooled human hepatocytes for 3-h to simulate liver metabolism. Incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) in full-scan and data-dependent MS/MS acquisition modes to capture the signal of all metabolites and their fragmentation pattern in two injections, i.e., in positive- and negative-ionization modes. LC-HRMS/MS data were mined with Compound Discoverer (Thermo Scientific) using a targeted/non-targeted approach to identify expected and unexpected metabolites. After excluding duplicates and putative metabolites with a molecular mass lower than 100 Da, 12 first-generation and 28 subsequent second-generation metabolites were predicted in silico; major reactions were O-sulfation, O-glucuronidation, N-dealkylation, and hydroxylation, N-oxidation, and carboxylation at the N-alkyl chain. 4-OH-MPT signal intensity decreased to 47% after 3-h incubation with hepatocytes. Seven metabolites were produced through N-demethylation and N-oxidation at the alkyl side chain, and O-glucuronidation and O-sulfation at the indole ring; 4-OH-MPT-glucuronide was the metabolite with the most intense signal. These results were consistent with the metabolic fate of structural analogues. The LC gradient was purposefully long and gradual to separate potential isomers. For this reason, O-glucuronides presented a broad shoulder peak, maybe indicating the resolution of two conformers of the same metabolites. 4-OH-MPT-N-oxide and 4-hydroxy-propyltryptamine (4-OH-PT) are proposed as metabolite biomarkers of 4-OH-MPT consumption after glucuronide and sulfate hydrolysis to increase 4-OH-MPT detection capabilities. Alternatively, 4-OH-MPT-glucuronide is suggested as an additional biomarker of 4-OH-MPT intake when hydrolysis is not performed. Unfortunately, these metabolites may not be specific to 4-OH-MPT. There are currently no data on the metabolism of structural analogues 4-hydroxy-N,N-dipropyltryptamine (4-OH-DPT), 4-acetoxy-N,N-dipropyltryptamine (4-AcO-DPT), and 4-acetoxy-N,N-methylpropyltryptamine (4-AcO-MPT) that potentially could produce similar metabolites. However, 4-OH-PT might be produced by 4-OH-DPT N-depropylation and 4-AcO-DPT ester hydrolysis and N-depropylation, making 4-OH-MPT-N-oxide crucial to document 4-OH-MPT consumption. More importantly, 4-AcO-MPT and 4-OH-MPT might produce the same major metabolites, considering the reactivity of the ester bond in 4-AcO-MPT. Therefore, the distinction between 4-OH-MPT and 4-AcO-MPT consumption may not be possible without detecting 4-AcO-MPT. The detection of tryptamine metabolites in biological matrices is crucial to document consumption in clinical and forensic settings. 4-OH-MPT-glucuronide, 4-OH-MPT-N-oxide, and 4-OH-PT are metabolite biomarkers of 4-OH-MPT consumption; however, research on the metabolism of other tryptamine structural analogues is necessary to evaluate the specificity of these metabolites to identify 4-OH-MPT only.

Integrating UPLC-HR-MS/MS, Network Pharmacology, and Experimental Validation to Uncover the Mechanisms of Jin'gan Capsules against Breast Cancer.

In the theory of traditional Chinese medicine (TCM), “liver-qi” stagnation and heat-induced toxicity represent the main etiologies of breast cancer. Recently, several TCMs with heat-clearing and detoxification efficacy have shown inhibitory effects on breast cancer. Jin’gan capsules (JGCs), initially approved to treat colds in China, are a heat-clearing and detoxification TCM formula. However, the anticancer activity of JGCs against breast cancer and its underlying mechanisms remain unclear. First, we assessed the antiproliferative activity of JGCs in breast cancer cell lines and evaluated their effects on cell apoptosis and the cell cycle by flow cytometry. Furthermore, we identified the potential bioactive components of JGCs and their corresponding target genes and constructed a bioactive compound–target interaction network by ultra-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HR-MS/MS) and network pharmacology analysis. Finally, the underlying mechanism was investigated through gene function enrichment analysis and experimental validation. We found that JGCs significantly inhibited breast cancer cell growth with IC50 values of 0.56 ± 0.03, 0.16 ± 0.03, and 0.94 ± 0.09 mg/mL for MDA-MB-231, MDA-MB-468, and MCF-7, respectively. In addition, JGC treatment dramatically induced apoptosis and S phase cell cycle arrest in breast cancer cells. Western blot analysis confirmed that JGCs could regulate the protein levels of apoptosis- and cell cycle-related genes. Utilizing UPLC-HR-MS/MS analysis and network pharmacology, we identified 7 potential bioactive ingredients in JGCs and 116 antibreast cancer targets. Functional enrichment analysis indicated that the antitumor effects of JGCs were strongly associated with apoptosis and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Western blot analysis validated that JGC treatment markedly decreased the expression levels of p-JAK2, p-STAT3, and STAT3. Our findings suggest that JGCs suppress breast cancer cell proliferation and induce cell cycle arrest and apoptosis partly by inhibiting the JAK2/STAT3 signaling pathway, highlighting JGCs as a potential therapeutic candidate against breast cancer.

Characterization and determination of benvitimod, an unknown risk substance in cosmetics, using nuclear magnetic resonance spectroscopy and HPLC-MS/MS.

Ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry, combined with preparative chromatography and nuclear magnetic resonance spectroscopy, a new method for identifying unknown risk substance structures in cosmetics has been established. Moreover, HPLC-MS/MS was developed for the determination of benvitimod in cosmetics. The sample was collected in ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry, and the molecular formula of the unknown was obtained as C17 H18 O2 . After preparative chromatography enrichment and purification, the enriched compound was scanned by nuclear magnetic resonance spectroscopy, and the chemical structure of the unknown compound was confirmed to be benvitimod. Subsequently, the separation was determined in multiple reaction monitoring mode. The results showed that the linearity of benvitimod was good in the range of 1-100 μg/L with a correlation coefficient r2 >0.999; the limits of detection and quantification were 0.02 mg/kg and 0.067mg/kg; the precision and stability were good; and the average recoveries were 104.2%, 108.2%, and 108.7% for low, medium, and high spiked concentrations, respectively. Forty batches of cosmetics were screened, of which two batches were detected with the illegal addition of benvitimod at 2.48 and 3.13 g/kg. The method effectively solved the loopholes in regulation and provided a research basis for the qualitative identification of structurally unknown compounds in cosmetics.

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome.

Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

Elucidation of in Vitro Chlorinated Tyrosine Adducts in Blood Plasma as Selective Biomarkers of Chlorine Exposure.

Chlorine is a widely available industrial chemical and involved in a substantial number of cases of poisoning. It has also been used as a chemical warfare agent in military conflicts. To enable forensic verification, the persistent biomarkers 3-chlorotyrosine and 3,5-dichlorotyrosine in biomedical samples could be detected. An important shortfall of these biomarkers, however, is the relatively high incidence of elevated levels of chlorinated tyrosine residues in individuals with inflammatory diseases who have not been exposed to chlorine. Therefore, more reliable biomarkers are necessary to distinguish between endogenous formation and exogeneous exposure. The present study aims to develop a novel diagnostic tool for identifying site-specific chlorinated peptides as a more unambiguous indicator of exogeneous chlorine exposure. Human blood plasma was exposed in vitro to various chlorine concentrations, and the plasma proteins were subsequently digested by pronase, trypsin, or pepsin. After sample preparation, the digests were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) and liquid chromatography high-resolution tandem mass spectrometry (LC–HRMS/MS). In line with other studies, low levels of 3-chlorotyrosine and 3,5-dichlorotyrosine were found in blank plasma samples in this study. Therefore, 50 site-specific biomarkers were identified, which could be used as more unambiguous biomarkers for chlorine exposure. Chlorination of the peptides TY*ETTLEK, Y*KPGQTVK, Y*QQKPGQAPR, HY*EGSTVPEK, and Y*LY*EIAR could already be detected at moderate in vitro chlorine exposure levels. In addition, the latter two peptides were found to have dichlorinated fragments. Especially, Y*LY*EIAR, with a distinct chlorination pattern in the MS spectra, could potentially be used to differentiate exogeneous exposure from endogenous causes as other studies reported that this part of human serum albumin is nitrated rather than chlorinated under physiological conditions. In conclusion, trypsin digestion combined with high-resolution MS analysis of chlorinated peptides could constitute a valuable technique for the forensic verification of exposure to chlorine.

Unveiling the Phytochemical Profile and Biological Potential of Five Artemisia Species.

The Artemisia L. genus comprises over 500 species with important medicinal and economic attributes. Our study aimed at providing a comprehensive metabolite profiling and bioactivity assessment of five Artemisia species collected from northeastern Romania (A. absinthium L., A. annua L., A. austriaca Jacq., A. pontica L. and A. vulgaris L.). Liquid chromatography–tandem high-resolution mass spectrometry (LC-HRMS/MS) analysis of methanol and chloroform extracts obtained from the roots and aerial parts of the plants led to the identification of 15 phenolic acids (mostly hydroxycinnamic acid derivatives), 26 flavonoids (poly-hydroxylated/poly-methoxylated flavone derivatives, present only in the aerial parts), 14 sesquiterpene lactones, 3 coumarins, 1 lignan and 7 fatty acids. Clustered image map (CIM) analysis of the phytochemical profiles revealed that A. annua was similar to A. absinthium and that A. pontica was similar to A. austriaca, whereas A. vulgaris represented a cluster of its own. Correlated with their total phenolic contents, the methanol extracts from both parts of the plants showed the highest antioxidant effects, as assessed by the DPPH and ABTS radical scavenging, CUPRAC, FRAP and total antioxidant capacity methods. Artemisia extracts proved to be promising sources of enzyme inhibitory agents, with the methanol aerial part extracts being the most active samples against acetylcholinesterase and glucosidase. All Artemisia samples displayed good antibacterial effects against Mycobacterium tuberculosis H37Ra, with MIC values of 64–256 mg/L. In conclusion, the investigated Artemisia species proved to be rich sources of bioactives endowed with antioxidant, enzyme inhibitory and anti-mycobacterial properties.

Comparison of methods for quantitative analysis of ranibizumab and bevacizumab in human plasma using various bioanalytical techniques, including microfluidic immunoassay, triple quadrupole, and high-resolution liquid chromatography-tandem mass spectrometry approaches

With the ever-growing abundance of complex therapeutic proteins reaching clinical trials and post-marketing, it is vital to develop highly accurate and robust bioanalytical methods for their quantitative analysis in matrices, to support clinical trial data as well as therapeutic drug monitoring. In bioanalysis, proteins have traditionally been evaluated using ligand binding assays (LBAs). However, in recent years, bottom-up LC-MS/MS methods have begun to gain recognition as an alternative to LBAs in situations where either there is a desire to reduce lengthy development times, or where selectivity issues prevent the immunoassay from reaching the desired outcome. In our study, a microfluidic immunoassay was compared to two bottom-up LC-MS/MS methods, including triple quadrupole and high-resolution mass spectrometry methods. The methods were designed to quantitatively analyze a monoclonal antibody, bevacizumab, and its related fab fragment, ranibizumab, in human plasma after intravitreal administration. All three methods were validated (or cross-validated) according to the 2018 Food and Drug Administration (FDA) guidance, and were then compared by quantitating eighteen patient samples on each platform. The concentrations values obtained from each method were compared using percent variability, as well as Bland-Altman and Pearson Correlation plots, to determine agreeability and linear correlation between methods. Based on the results of the validations and comparison studies, all three methods aligned well with each other. However, the LC-MS/MS methods were able to achieve significantly improve sensitivity, with a lower limit of quantitation (LLOQ) of 0.300 ng/mL, compared to 6.00 ng/mL for the LBA, due to the reduction of interferences at lower concentrations using the LC-MS/MS technique (increased selectivity). Therefore, for this specific study, we were able to establish the correlation between methods, while also demonstrating increased value in using LC-MS/MS as an alternative approach to LBAs in bioanalysis.

Soil-derived Streptomyces sp. GMR22 producing antibiofilm activity against Candida albicans: bioassay, untargeted LC-HRMS, and gene cluster analysis

Biofilm-forming fungi, Candida albicans, are currently a serious problem in infectious disease cases. Soil bacteria Streptomyces sp. GMR22 have a large genome size and antifungal metabolites against C. albicans, but its potential antibiofilm activity is not clearly defined. The aims of this study were to determine the antibiofilm activity of GMR22 against C. albicans, identify the main constituents of active extracts, and investigate the biosynthesis gene clusters encoding the enzymes related to metabolism pathways. Antifungal and antibiofilm measurements were performed using in vitro assays on C. albicans ATCC 10231. Main constituents of active extracts were analyzed using untargeted Liquid Chromatography tandem High-Resolution Mass Spectrometry (LC-HRMS). RAST software was applied to investigate the gene clusters of the biosynthesis pathways based on whole genome sequences. Chloroform extract of GMR22 has antifungal and antibiofilm properties at 13–420 μg/mL with palmitic acid (C16H32O2, 273.27028 Da), a saturated fatty acid as a major constituent (42.74). Streptomyces sp. GMR22 has 53 subsystems related to fatty acids biosynthesis (Fab) FAS II. The Kyoto Encyclopedia of Gene and Genome map of Fab revealed 10 of 21 (47.6%) gene clusters encode enzymes related to Fab. There were six gene clusters encoding the enzymes related to the hexadecenoic acid (palmitic acid) biosynthesis pathways: 6.4.12; FabD, FabH, FabF, FabG, FabI and 1.14.192. Each enzyme was encoded by 3–14 genes. These results confirmed that soil Streptomyces sp. GMR22 bacterium has remarkable biotechnological potential by producing fatty acids which are mostly palmitic acid as an active antibiofilm agent against C. albicans.

Proteomic Analysis of Serum Lysine Acetylation in Uyghur Patients With T2DM

Lysine acetylation is a reversible modification process after protein translation, which plays a key regulatory role in various metabolic diseases such as diabetes. The prevalence of type 2 diabetes mellitus (T2DM) in the Uyghur population is high, but the acetylation status of proteomics in Uyghur with T2DM is still unclear. Herein, we performed a quantitative proteomic study of lysine acetylation in T2DM patients using Tandem Mass Tags (TMTs) labeling, acetylation enrichment techniques, and high-resolution liquid chromatography-tandem mass spectrometry. We quantified 422 acetylation sites on 120 proteins, of which 347 sites of 103 proteins contained quantitative information. Compared with the control, we found that a total of eight acetylated sites within proteins were significantly differentially expressed with three upregulated and five downregulated, including histones H4 and H3.3C. Meanwhile, we completed bioinformatics analysis, including protein annotation, functional classification, functional enrichment, and cluster analysis, based on functional enrichment. In addition, the mRNA (ApoB-100, histones H4 and H3.3C) and protein (histones H4 and H3.3C) levels were verified through 60 samples. Besides, we also performed histone H4 chromatin immunoprecipitation analysis at the level of INS-1 cells. These could be potentially useful markers for the prediction of prediabetes and also provided a basis for the pathogenesis of T2DM.