Project description:Although the benefits of reduction of the size of reversed phase particles are established to provide increased sequencing depth and improved chromatography in LCMS experiments, the wide-scale adoption of optimally sized small particles in reversed-phase columns has been hampered by the necessity for specialized equipment such as ultra-high pressure liquid chromatography or a customized column heating apparatus. Here, we introduce a new strategy to routinely fabricate a 50 cm-long, 1.9 µm particle C18 column and extensively characterize the performance of this column. This column was packed under 100 Bar and routinely utilized on a standard quarternary HPLC at pressures below 300 Bar. Expanding the depth of sequencing of peptides that show a statistically significant quantitative change arising from a biological stimulation is critical. Compared with traditional C18 columns packed with 3 µm particles, the column with the 1.9 µm particles operated with a standard HPLC could detect 330% more peptides with statistically significant changes from differentially stimulated T cells. This improved column fabrication methodology provides an inexpensive improvement for single-run LC-MS/MS analysis to optimize sequencing depth, dynamic range, sensitivity, and reproducibility. This study also highlights the importance of the statistical analysis of quantitative proteomic data instead of a sole focus on peptide spectrum match yields.
Project description:Vendor raw LC-MS/MS data and supporting files for the study "Leucine supplementation modulates lipid metabolism in STZ-induced type 1 diabetic mice". Waters Xevo G2-XS QTOF with ACQUITY BEH C18 column in positive ESI mode. Structured metadata, and the full LCMS protocol are included. Raw vendor files are provided as 16 multi-part 7z archives: RAW files.7z.001 ... RAW files.7z.016. Please see README_reassembly.txt for reassembly.
Project description:Human serum samples from Johns Hopkins University. LCMS run on 05-09-2023. Polar C18 column in positive mode. This project is about the chagas disease progression. Four groups in this dataset: infected-progressed, infected-non-progressed, uninfected-progressed, uninfected-non-progressed. We want to compare the progressed and non-progressed in infected groups to see the changes of metabolites related to the disease progression.
Project description:Introduction: Atherosclerosis can trigger various cardiovascular and cerebrovascular diseases with complex pathogenesis. Macrophage proliferation, inflammatory responses, and lipid phagocytosis, which induce foam cell formation and accumulation, are critical in the development of early atherosclerotic lesions. The role of 3-Hydroxystearic acid (C18-3OH), a recently identified gut microbiota-derived metabolite, in atherosclerosis has not yet been clarified. This study aimed to investigate the role of the ALKBH5/PAX-8/ABCA1 pathway in C18-3OH-mediated regulation of macrophage cholesterol efflux and atherosclerosis and explore novel mechanisms of ABCA1 regulation from the perspective of m6A modification. Methods: RT-qPCR and Western blotting were used to detect gene and protein expression, respectively. ChIP-Seq was used to screen PAX-8 target genes, and ChIP-qPCR was used to validate PAX-8 binding to ABCA1. The SRAMP platform was used to predict m6A modification sites in PAX-8 mRNA sequences. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was used to measure m6A modification levels of PAX-8 mRNA in foam cells. UHPLC-OEMS untargeted metabolomics were used to analyze differential fatty acid metabolites in an atherosclerotic mouse model. Specific kits were used to detect serum liver function markers (aspartate transaminase, AST; alanine aminotransferase, ALT), renal function markers (serum creatinine, Scr; blood urea nitrogen, BUN), and lipid profiles (HDL-C, TG, LDL-C, TC). Aortic sinus sections were prepared, and H&E, Oil Red O, and Masson staining were used to evaluate atherosclerotic plaques. Results: The results demonstrated that C18-3OH promoted cholesterol efflux in foam cells and alleviated lipid accumulation by upregulating ABCA1 expression. C18-3OH inhibited ALKBH5, increased PAX-8 mRNA m6A modification and PAX-8 expression, and upregulated ABCA1 to enhance cholesterol efflux. Serum metabolomics revealed reduced C18-3OH levels in high-fat diet-fed apoE-/- atherosclerotic mice. C18-3OH suppressed aortic ALKBH5 expression, elevated m6A modification of PAX-8 mRNA, and increased PAX-8 and ABCA1 expression. Furthermore, C18-3OH improved lipid metabolism and reduced the atherosclerotic plaque area in apoE-/- mice. Discussion: This study clarifies the impact and mechanisms of gut microbiota-derived C18-3OH on atherosclerosis progression, providing novel strategies for the precise prevention and treatment of atherosclerosis.
Project description:UHPLC-HRMS/MS data of murine colon tissue in positive and negative mode. Metabolomics. Reverse phase chromatography using F5 column. Iterative DDA data collected using AcquireX procedure on Thermo MS.
Project description:In current work, we compared the separation of HeLa samples using two different trap columns for sample trapping, desalting, and preconcentration. The two columns have different types of the C18 stationary phase: a) a conventional packed PepMap C18 trap-column (Thermo Fischer Scientific), and b) the superficially porous silicon pillars μPAC C18 trap-column (PharmaFluidics)
Project description:This project applies proteomic stable isotope probing (proteomic SIP) to evaluate detection and quantification of 13C‑labeled E. coli peptides within a complex mouse fecal microbiome background. Proteins were extracted, digested, and quantified before preparing spike‑in mixtures. Mouse fecal peptides (2 µg) were combined with E. coli peptides at 10:1 or 100:1 ratios using defined 13C labeling levels (2%, 5%, 50%), each in triplicate. Peptides were analyzed by nanoLC–MS/MS on an XSelect CSH C18 column coupled to an Orbitrap Fusion Tribrid mass spectrometer in data‑dependent acquisition mode. The dataset enables controlled evaluation of isotopic incorporation, quantitative behavior, and sensitivity of proteomic SIP in microbiome samples.
Project description:MIADB: a cumulative collection of 172 tandem mass spectrometry (MS/MS) of a vast array of monoterpene indole alkaloids. Samples were analyzed using an Agilent LC-MS system composed of an Agilent 1260 Infinity HPLC coupled to an Agilent 6530 ESI-Q-TOF-MS operating in positive mode. A Sunfire analytical C18 column (150 × 2.1 mm; i.d. 3.5 μm, Waters) was used, with a flow rate of 250 μL/min and a linear gradient from 5% B (A: H2O + 0.1% formic acid, B: MeOH) to 100% B over 30 min. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 10 L/min. The divert valve was set to waste for the first 3 min. There were four scan events: positive MS, window from m/z 100−1200, then three data-dependent MS/MS scans of the first, second, and third most intense ions from the first scan event. MS/MS settings were three fixed collision energies (30, 50, and 70 eV), default charge of 1, minimum intensity of 5000 counts, and isolation width of m/z 2. In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z 121.050873) and the hexakis(1H,1H,3H-tetrafluoropropoxy)-phosphazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used as internal lock masses. Full scans were acquired at a resolution of 11 000 (at m/z 922). A permanent MS/MS exclusion list criterion was set to prevent oversampling of the internal calibrant.