Project description:Proteomics and transcriptomics data of tomato fruits (Solanum lycopersicum L. var. Moneymaker) at 9 developmental stages were used to calculate with a mathematical model the rate constants of synthesis and degradation for over 1,000 proteins. Proteome and transcriptome were extracted from the pericarp tissue and analyzed using label-free LC-MS/MS (Orbitrap Q-Exactive) and RNA Sequencing (Illumina), respectively. Absolute quantification of transcriptome has been obtained by spiking-in internal standard before total-RNA extraction. Absolute quantification of the proteome has been approximated using the "Total Protein" approach. An OD equation defining the changes of protein content has been used to determine the synthesis and degradation rate constants (day -1). Almost 2,400 transcript-protein pairs were identified and the translation and degradation rate constants were determined for more than a thousand proteins. The model predicted median values of about 2 min for the translation and a lifetime of approximately 11 days. Proteins involved in protein synthesis had higher ks and kd values, indicating that the protein machinery is particularly flexible. None sequenced-based features were found that could be used to predict these rate constants.
Project description:We profiled polar metabolites across multiple organs and fluids from C57BL/6NCrl (B6NCrl-MVP) wild-type mice at two ages (16 and 92 weeks), including both sexes. Samples spanned the nervous (hippocampus, cerebrospinal fluid), connective (brown, subcutaneous, and visceral/abdominal fat), digestive (stomach, duodenum, jejunum, ileum, cecum, colon, liver, gallbladder), immune (spleen, thymus), reproductive (testes, uterus), excretory (kidney, bladder), endocrine (pancreas), cardiovascular (heart), muscular (quadriceps), and respiratory (lung) systems, plus biofluids (plasma, urine) and digestive products (feces). Polar metabolomics was performed by LC-MS/MS on a Thermo Scientific Q Exactive HF-X Quadrupole-Orbitrap coupled to a Vanquish UHPLC in positive mode. Metabolites were separated on a Waters ACQUITY Premier BEH Amide column (50 x 2.1 mm, 1.7 um) prior to high-resolution MS acquisition.
Project description:<p>Irinotecan (CPT-11) is widely used for colorectal cancer treatment, with delayed-onset diarrhea as its primary side effect. Xiao-Chai-Hu-Tang (XCHT) has been clinically observed to alleviate diarrhea, but its mechanisms remain unclear. This study aimed to explore the underlying regulatory mechanisms through which XCHT alleviated CPT-11-induced diarrhea. The chemical components of XCHT were detected using UHPLC-Q-Exactive Orbitrap-MS, and 12 active components were quantified via UHPLC-QQQ-MS/MS to ensure quality stability. CPT-11-induced diarrheal mice were established to evaluate the effects of XCHT. Differential metabolites in liver and intestinal tissues among control, CPT-11, and XCHT mice was analyzed by metabolomics, followed by molecular network analysis using IPA. The bile acids were quantified by using the UHPLC-QQQ-MS/MS method. Proteomics of colon tissues identified differentially expressed proteins, with functional enrichment conducted via GO and KEGG. Western blot and flow cytometry were used to validate potential biopathway. XCHT primarily contained flavonoids, carbohydrates and glycosides, and terpenoids. Twelve active components remained stable throughout the experiment. XCHT significantly improved diarrhea symptoms. Metabolomics revealed 133 and 72 differential metabolites in the liver and intestine, respectively, and linked to bile acid metabolism. Further targeted bile acid analysis revealed that CPT-11 reduced hepatic bile acid synthesis and uptake, but increased biliary excretion. Significantly elevated bile acid levels were observed in the gallbladder of CPT-11-treated mice. Furthermore, intestinal bile acid reabsorption was impaired with an 81% decrease in total bile acids and reduced uptake/efflux, resulting in a 56% decline in circulatory bile acids, and 40% increase in colonic accumulation. Proteomics identified 86 differentially expressed proteins in colon tissues, with significant enrichment in ‘Focal adhesion’. Integrated analysis and validation studies suggested the TGR5-FAK-RhoA/ROCK signaling axis as the crucial pathway through which XCHT alleviated diarrhea. These findings provide novel insights into the pharmacological mechanisms of XCHT and suggest potential therapeutic strategies targeting bile acid metabolism for chemotherapy-induced diarrhea.</p>
Project description:To identify protein binding partners of endogenous RBMS1, it was immunoprecipicated in triplicate, vs. IgG control, from SW480 colon adenocarcinoma cells, and subjected to MS analysis on an Q-exactive plus Orbitrap mass spectrometer. Raw files 13, 14, and 15 correspond to biological replicate IPs of RBMS1, and Raw files 16, 17, and 18 correspond to biological replicate IPs of IgG control.
Project description:Background: Fangji Dihuang formulation (FJDHF) is a well-known Traditional Chinese Medicine (TCM) formula with a reported clinical therapeutic effect in the treatment of inflammatory skin diseases. However, there is a lack of pharmacological research on its anti-atopic dermatitis (AD) activity. Methods: To investigate the potential anti-AD activity of FJDHF, DNCB was used to induce AD-like skin inflammation in the back of mice. Following successful modeling, the mice were administered FJDHF orally. The extent of the inflammatory skin lesions was recorded at day 4, 7, 14 and 28. UHPLC-Q-Exactive Orbitrap MS was used to identify and match the compounds present in FJDHF with ITCM, TCMIP and TCMSID. In silico predictions of potential target proteins of the identified compounds were obtained from SwishTargetPrediction, ITCM and TargetNet databases. AD-related genes were identified from GSE32924 data set, and FJDHF anti-AD hub genes were identified by MCODE algorithm. ClueGo enrichment analysis was employed to identify the core pathway of FJDHF's anti-AD effect. To further investigate the anti-AD effect of FJDHF, single-cell RNA sequencing data set (GSE148196) from AD patients was analyzed to determine the target cells and signaling pathways of FJDHF in AD. Finally, rt-PCR, flow cytometry, and mouse back skin RNA sequencing were utilized to validate our findings. Results: FJDHF was found to be effective in improving the degree of the AD-like lesions in the mice. Network pharmacological analysis revealed the core pathway of FJDHF to be the IL-17 signaling pathway, which is interactively associated with cytokines. Single-cell RNA sequencing analysis suggested that FJDHF may play an anti-AD role by influencing dendritic cells. Flow cytometry and rt-PCR results showed that FJDHF can reduce the influence of AD sample of IL-4, IFN-γ and the expression of IL-17. The RNA sequencing of mouse back skin also confirmed our conclusion. Conclusion: FJDHF may inhibit DNCB-induced AD-like skin inflammation in mice by inhibiting the IL-17 signaling pathway. Thus, FJDHF can be considered as a potential therapeutic agent for AD.
Project description:We profiled lipids across multiple organs and biofluids from C57BL/6NCrl (B6NCrl-MVP) wild-type mice at two ages (16 and 92 weeks), including both sexes. Samples spanned the nervous (hippocampus, cerebrospinal fluid), connective (brown, subcutaneous, and visceral/abdominal fat), digestive (stomach, duodenum, jejunum, ileum, cecum, colon, liver, gallbladder), immune (spleen, thymus), reproductive (testes, uterus), excretory (kidney, bladder), endocrine (pancreas), cardiovascular (heart), muscular (quadriceps), and respiratory (lung) systems, plus biofluids (plasma, urine) and digestive products (feces). Lipidomics was performed by LC-MS/MS on a Thermo Scientific Orbitrap Exploris 240 coupled to a Vanquish UHPLC in both positive and negative ion modes. Analytes were separated on a Waters ACQUITY Premier BEH C18 column (50 x 2.1 mm, 1.7 um) prior to high-resolution MS acquisition.
Project description:E-Jiao is a traditional Chinese medicine. In this project, we used Q Exactive Orbitrap Plus to identify the peptides from E-Jiao. We also used the QE Plus to investigate the E-Jiao peptides in rat blood.