A UPLC-Q-TOF/MS-based plasma metabolomics approach reveals the mechanism of Compound Kushen Injection-based intervention against non-small cell lung cancer in Lewis tumor-bearing mice

BackgroundCompound Kushen Injection (CKI), a well-known Chinese Medicine preparation, has been used to treat non-small cell lung cancer (NSCLC) for more than 15 years, and its clinical curative effect is considered to be beneficial.Hypothesis/PurposeThis study was designed to evaluate the effects and underlying mechanisms of CKI against NSCLC using an ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based plasma metabolomics approach.Methods4′,6-diamidino-2-phenylindole (DAPI) staining and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay were employed to assess apoptosis and the viability of A549 cells with and without CKI treatment. The weight/volume of Lewis lung carcinoma (LLC) sarcomas and histopathological examinations were used to evaluate the anti-tumor effects of CKI against NSCLC. A UPLC-Q-TOF/MS method combined with multivariate data analysis was developed to characterize metabolomic fingerprinting and to screen functional biomarkers that are linked to the CKI treatment of LLC mice, and then metabolic pathway analysis was used to investigate the therapeutic mechanism of CKI.ResultsDAPI staining and MTT dye reduction assays indicated that CKI-induced apoptosis and inhibited the proliferation of A549 cells, respectively, in a concentration-dependent manner. The sarcoma volumes and weights in LLC tumor-bearing mice in CKI-dosed groups were significantly lower than those in a model group, which was treated with physiological saline. Histopathological analysis of sections of sarcomas and left pulmonary lobes indicated that CKI exerts an ameliorative effect against LLC. Fourteen functional biomarkers that are related to the therapeutic effects of CKI on LLC were screened and identified using a metabolomics study. Analysis of metabolic pathways revealed that the therapeutic effects of CKI on LLC mainly involved glycerophospholipid metabolism, amino acid metabolism and sphingolipid metabolism. As glycerophospholipid metabolism is a crucial feature of cancer-specific metabolism, the enzymes that are involved in 1-acyl-sn-glycero-3-phosphoinositol biosynthesis were further evaluated. Western blotting results indicated that CKI modulated the abnormal biosynthesis pathway of 1-acyl-sn-glycero-3-phosphoinositol by activation of cytidine diphosphate-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and cytosolic phospholipase A2 (cPLA2), and by inhibition of lysophosphatidic acid acyltransferase gamma (AGPAT3).ConclusionThis study demonstrated that CKI has a favorable anti-tumor effect and that a UPLC-Q-TOF/MS-based metabolomics method in conjunction with further verifications at the biochemical level is a promising approach for investigating its underlying mechanisms.     

State-of-the-art non-targeted metabolomics in the study of chronic kidney disease

Here we report a metabolomics discovery study conducted on blood serum samples of patients in different stages of chronic kidney disease (CKD). Metabolites were monitored on a quality controlled holistic platform combining reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry in both negative and positive ionization mode and gas chromatography coupled to quadrupole mass spectrometry. A substantial portion of the serum metabolome was thereby covered. Eighty-five metabolites were shown to evolve with CKD progression of which 43 metabolites were a confirmation of earlier reported uremic retention solutes and/or uremic toxins. Thirty-one unique metabolites were revealed which were increasing significantly throughout CKD progression, by a factor surpassing the level observed for creatinine, the currently used biomarker for kidney function. Additionally, 11 unique metabolites showed a decreasing trend.     

Analytical strategies for LC-MS-based targeted metabolomics

Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple quadrupole mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.     

麦冬提取物治疗2型糖尿病小鼠的血清代谢组学研究*

目的:采用代谢组学方法研究麦冬对2型糖尿病(T2DM)小鼠内源性代谢物的影响。方法: 腹腔注射链脲佐菌素建立T2DM小鼠模型。连续4周灌胃给予麦冬水提物后,检测血清中糖尿病相关指标,且以超高效液相色谱-飞行时间质谱联用(UPLC-Q/TOF-MS)技术,研究麦冬对T2DM小鼠血清中代谢物质的影响,分析相关代谢通路。结果: 与正常对照组相比,模型组血清的空腹葡萄糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇含量显著升高,高密度脂蛋白胆固醇含量显著降低;灌胃给予麦冬水提物后,T2DM小鼠血清中以上指标均明显改善。代谢组学结果显示,正常组与模型组共有43个差异代谢物,富集于18条通路;麦冬提取物明显降低T2DM小鼠的甘油酸、丙二酸半醛和4-羟基苯基丙酮酸含量,差异代谢物富集于泛醌和其他萜类醌生物合成代谢等7条通路。结论: 麦冬水提物具有降低T2DM小鼠血糖和血脂的药效作用,且可能与泛醌和其他萜类醌生物合成等通路有关。     

LC-MS-based untargeted metabolomics reveals the mechanism underlying prostate damage in a type 2 diabetes mouse model

Type 2 diabetes mellitus (T2DM) can cause prostate damage and affect male reproductive function, but the underlying mechanisms are not completely understood. In this study, we used liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics to identify endogenous metabolites in the prostate of a T2DM mouse model. The selected endogenous metabolites were then subjected to bioinformatics analysis and metabolic pathway studies to understand their role in the development of T2DM-induced prostate damage. We used male homozygous BTBR ob/ob mice (n = 12) and BTBR WT mice (n = 11) in this study. We monitored changes in blood glucose, body weight, prostate weight, and prostate index, as well as performed hematoxylin and eosin (H&E) staining and observed that the prostate of the BTBR ob/ob was damaged. We then used ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for metabolomics analysis. The stability of the model was validated using principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Using variable importance in projection (VIP) > 1, false discovery rate (FDR) < 0.05, and coefficient of variation (CV) < 30 as criteria, a total of 149 differential metabolites (62 upregulated and 87 downregulated) were identified between the prostates of the two groups of mice. Topological pathway analysis showed that these differential metabolites were mainly involved in sphingolipid (SP) and glycerophospholipid (GP) metabolism. In conclusion, our study not only emphasizes the damage caused by T2DM to the prostate but also provides new insights into the potential mechanisms of T2DM-induced male reproductive dysfunction.     

Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation.     

Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins

We performed a proteomic analysis of monocytes primed by lipopolysaccharide (LPS) in vitro, using two-dimensional gels stained with Coomassie blue. We found 16 proteins of approximately 500 detected that either increased or decreased in abundance as a result of priming by LPS (14 with P 相似文献   

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge on CO₂ signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO₂ using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performance LC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevated CO₂ level. Most metabolites increased under elevated CO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO₂ treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO₂ signaling mutants, we discovered that CO₂‐induced stomatal closure is mediated by JA signaling.     

Lipopolysaccharide-responsive phosphoproteins in Nicotiana tabacum cells.

Mounting evidence is merging to affirm the effectiveness of bacterial lipopolysaccharides (LPS) as biological control agents, inducers of innate immunity, and to stimulate/potentiate the development of defense responses in plants through protein phosphorylation-mediated signal perception/transduction responses. In vivo labeling of protein phosphorylation events during signal transduction indicated the rapid phosphorylation of several proteins. Substantial differences and de novo LPS-induced phosphorylation were also observed with two-dimensional analysis. In this study, qualitative and quantitative changes in phosphoproteins of Nicotiana tabacum suspension cells during elicitation by LPS from the Gram-negative bacteria, Burkholderia cepacia, were analyzed using two-dimensional electrophoresis in combination with a phosphoprotein-specific gel stain. Trypsin digested phosphoproteins were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and nano-electrospray-ionization liquid chromatography tandem mass spectrometry (nano-ESI-LC/MS/MS). A total of 27 phosphoproteins were identified from 23 excised gel spots. The identified phosphoproteins indicate that LPS(B.cep)-induced signal perception/transduction involves G-protein coupled receptor signaling, Ca(2+)/calmodulin-dependent signaling pathways, H(+)-ATPase regulation of intracellular pH, thioredoxin-mediated signaling and phosphorylation of 14-3-3 regulatory proteins. Other targets of LPS(B.cep)-responsive phosphorylation included NTP pool maintenance, heat shock proteins, protein biosynthesis and chaperones as well as cytoskeletal tubulin. The results add novel insights into the biochemical process of LPS perception and resulting signal transduction.     

A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue

The revival of interest in cancer cell metabolism in recent years has prompted the need for quantitative analytical platforms for studying metabolites from in vivo sources. We implemented a quantitative polar metabolomics profiling platform using selected reaction monitoring with a 5500 QTRAP hybrid triple quadrupole mass spectrometer that covers all major metabolic pathways. The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze 258 metabolites (289 Q1/Q3 transitions) from a single 15-min liquid chromatography-mass spectrometry acquisition with a 3-ms dwell time and a 1.55-s duty cycle time. Previous platforms use more than one experiment to profile this number of metabolites from different ionization modes. The platform is compatible with polar metabolites from any biological source, including fresh tissues, cancer cells, bodily fluids and formalin-fixed paraffin-embedded tumor tissue. Relative quantification can be achieved without using internal standards, and integrated peak areas based on total ion current can be used for statistical analyses and pathway analyses across biological sample conditions. The procedure takes ～12 h from metabolite extraction to peak integration for a data set containing 15 total samples (～6 h for a single sample).     

Impact of a Transposon Insertion in phzF2 on the Specialized Metabolite Production and Interkingdom Interactions of Pseudomonas aeruginosa

In microbiology, gene disruption and subsequent experiments often center on phenotypic changes caused by one class of specialized metabolites (quorum sensors, virulence factors, or natural products), disregarding global downstream metabolic effects. With the recent development of mass spectrometry-based methods and technologies for microbial metabolomics investigations, it is now possible to visualize global production of diverse classes of microbial specialized metabolites simultaneously. Using imaging mass spectrometry (IMS) applied to the analysis of microbiology experiments, we can observe the effects of mutations, knockouts, insertions, and complementation on the interactive metabolome. In this study, a combination of IMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to visualize the impact on specialized metabolite production of a transposon insertion into a Pseudomonas aeruginosa phenazine biosynthetic gene, phzF2. The disruption of phenazine biosynthesis led to broad changes in specialized metabolite production, including loss of pyoverdine production. This shift in specialized metabolite production significantly alters the metabolic outcome of an interaction with Aspergillus fumigatus by influencing triacetylfusarinine production.     

Peptidomics of the locust corpora allata: identification of novel pyrokinins (-FXPRLamides)

The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.     

Metabolomics study of type 2 diabetes using ultra-performance LC-ESI/quadrupole-TOF high-definition MS coupled with pattern recognition methods

Type 2 diabetes (T2D), called the burden of the twenty-first century, is growing with an epidemic rate. Here, we explored the differences in metabolite concentrations between T2D patients and healthy volunteers. Metabolomics represents an emerging discipline concerned with comprehensive analysis of small molecule metabolites and provides a powerful approach to discover biomarkers in biological systems. The acquired data were analyzed by ultra-performance liquid chromatography–electrospray ionization/quadrupole time-of-flight high-definition mass spectrometry coupled with pattern recognition approach [principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)] to identify potential disease-specific biomarkers. PCA showed satisfactory clustering between patients and healthy volunteers. Biomarkers reflected the biochemical events associated with early stages of T2D which were observed in PLS-DA loading plots. These urinary differential metabolites, such as adiponectin, acylcarnitines, citric acid, kynurenic acid, 3-indoxyl sulfate, urate, and glucose, were identified involving several key metabolic pathways such as taurine and hypotaurine metabolism; cysteine and methionine metabolism; valine, leucine, and isoleucine biosynthesis metabolism, etc. Our data suggest that robust metabolomics has the potential as a noninvasive strategy to evaluate the early diagnosis of T2D patients and provides new insight into pathophysiologic mechanisms and may enhance the understanding of its cause of disease.     

Insight into the surfactin production of <Emphasis Type="Italic">Bacillus velezensis</Emphasis> B006 through metabolomics analysis

Bacillus velezensis B006 is a biocontrol agent which functions through effective colonization and surfactin production. To reveal the surfactin-producing mechanism, gas chromatography–mass spectrometry based untargeted metabolomics was performed to compare the metabolite profiles of strain B006 grown in industrial media M3 and M4. Based on the statistical and pathway topology analyses, a total of 31 metabolites with a fold change of less than ??1.0 were screened as the significantly altered metabolites, which distributed in 15 metabolic pathways. Fourteen amino acids involving in the metabolisms of alanine/aspartate/glutamate, glycine/serine/threonine, arginine/proline, glutathione/cysteine/methionine and valine/leucine/isoleucine as well as succinic acid in TCA cycle were identified to be the hub metabolites. Aminoacyl-tRNA biosynthesis, glycerolipid metabolism, and pantothenate/CoA biosynthesis also contributed to surfactin production. To the best of our knowledge, this study is the first to investigate the metabolic pathways of B. velezensis on surfactin production, and will benefit the optimization of commercial fermentation for higher surfactin yield.     

Metabolomics analysis of collagen-induced arthritis in rats and interventional effects of oral tolerance

A serum metabolomics method based on rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RRLC-Q-TOF-MS) was performed for a holistic evaluation of the metabolic changes of collagen-induced arthritis (CIA) in rats and to assess the interventional effects of type II collagen (CII) in this model. Partial least-squares-discriminant analysis (PLS-DA) was employed to study the metabolic profiling of CIA rats and control rats. Ten metabolites, namely, 12(S)-HHTrE, 12(S)-HEPE, PGE₂, TXB2, 12(S)-HETE, LysoPE(16:0), PE(O-18:0/0:0), Lyso-PE(18:2), Lyso-PE(20:4), and Lyso-PC(22:5) were identified as differential metabolites associated with the pathogenesis of CIA. These results suggested that dysregulation of the arachidonic acid (AA) and phospholipid metabolic networks is involved in the pathomechanism of CIA. Differential metabolomics and histopathological analyses demonstrated that CII inhibits the progress of arthritis. Furthermore, the therapeutic effects of CII on CIA may involve regulation of the disordered AA and phospholipid metabolic networks. This metabolomics study provides new insights into the pathogenesis of arthritis and, furthermore, indicates the potential mechanism underlying the significantly increased prevalence of metabolic syndrome, defined as a clustering of cardiovascular disease (CVD) risk factors, in arthritis patients.     

Comparative study of the LC-MS/MS and UPLC-MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC

The aim of this study was to develop and validate an analytical method to simultaneously determine European Union-regulated β-lactams (penicillins and cephalosporins) and quinolones in cow milk. The procedure involves a new solid phase extraction (SPE) to clean-up and pre-concentrate the three series of antibiotics before analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). LC-MS/MS and UPLC-MS/MS techniques were also compared. The method was validated according to the Directive 2002/657/EC and subsequently applied to 56 samples of raw cow milk supplied by the Laboratori Interprofessional Lleter de Catalunya (ALLIC) (Laboratori Interprofessional Lleter de Catalunya, Control Laboratory Interprofessional of Milk of Catalunya).     

Molecular signature of renal cell carcinoma by means of a multiplatform metabolomics analysis

Renal cell carcinoma (RCC) is a disease with no specific diagnostic method or treatment. Thus, the evaluation of novel diagnostic tools or treatment possibilities is essential. In this study, a multiplatform untargeted metabolomics analysis of urine was applied to search for a metabolic pattern specific for RCC, which could enable comprehensive assessment of its biochemical background. Thirty patients with diagnosed RCC and 29 healthy volunteers were involved in the first stage of the study. Initially, the utility of the application of the selected approach was checked for RCC with no differentiation for cancer subtypes. In the second stage, this approach was used to study clear cell renal cell carcinoma (ccRCC) in 38 ccRCC patients and 38 healthy volunteers. Three complementary analytical platforms were used: reversed-phase liquid chromatography coupled with time-of-flight mass spectrometry (RP-HPLC-TOF/MS), capillary electrophoresis coupled with time-of-flight mass spectrometry (CE-TOF/MS), and gas chromatography triple quadrupole mass spectrometry (GC-QqQ/MS). As a result of urine sample analyses, two panels of metabolites specific for RCC and ccRCC were selected. Disruptions in amino acid, lipid, purine, and pyrimidine metabolism, the TCA cycle and energetic processes were observed. The most interesting differences were observed for modified nucleosides. This is the first time that the levels of these compounds were found to be changed in RCC and ccRCC patients, providing a framework for further studies. Moreover, the application of the CE-MS technique enabled the determination of statistically significant changes in symmetric dimethylarginine (SDMA) in RCC.     

Spatial metabolomics using imaging mass spectrometry to identify the localization of asparaptine A in Asparagus officinalis

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis.