Project description:<p _msttexthash='64160037318' _msthash='272'>Ultra-high-performance liquid chromatography (UHPLC) was conducted using a Vanquish UHPLC system (Thermo Fisher), coupled with a Q Exactive™ HF/Q Exactive™ HF-X mass spectrometer for liquid chromatography-mass spectrometry (LC-MS) analysis. To ensure high resolution and effective analyte retention, a Hypesil Gold chromatographic column (100 × 2.1 mm, 1.9 µm) was used to separate the samples by hydrophilic interaction chromatography (HILIC). In both positive and negative electrospray ionization (ESI) modes, the chromatographic parameters were as follows: flow rate: 0.2 mL/min, column temperature: 40℃, mobile phase A = 0.1% formic acid, B = methanol. The separated metabolites were then analyzed via mass spectrometry, with a scanning range of m/z 100–1500. The MS/MS secondary scan was performed using a data-dependent acquisition mode, and QC samples were used to correct instrument drift and signal fluctuation. Subsequently, the raw LC-MS data files were processed using Compound Discoverer 3.3 (CD3.3; Thermo Fisher Scientific) (Cooper and Yang, 2024). Initially, each metabolite was screened, and the peak area was normalized using the first QC sample. In the subsequent steps, high-resolution molecular ion peaks and characteristic fragment ions were used to predict molecular formulas, and compared with the mzCloud, mzVault, and MassList databases to achieve accurate metabolite identification and relative quantification.</p>
Project description:M. hominis cells were grown in liquid medium supplied with arginine or thymidine as a carbon source. LC-MS analysis was performed on Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled with Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific), an untargeted label-free bottom-up proteomic strategy was used, DDA (Data Dependent Acquisition) approach. Protein identification and label-free quantification were performed with PEAKS software.
Project description:In this project, Q-Exactive HF(Thermo Fisher Scientific,San Jose,CA) was used to acquire mass spectrometry (MS) data for 7 samples in Data Independent Acquisition (DIA) mode. Quantification of peptides and proteins was performed using Spectronaut™ and MSstats software packages
Project description:An TMT-based quantitative crotonylome analysis was performed on the crotonylated proteins enriched from the chloroplast extracts of the normal and salt-treated wheat seedling leaves by using affinity purification and LC-MS/MS. The crotonylated peptides from wheat chloroplast extracts were enriched by using anti-crotonyllysine mouse mAb (Clone 4D5) antibody (PTM BioLabs, HangZhou, China PTM-501 and Nano LC-MS/MS was performed by using a Dionex rapid-separation liquid chromatography system interfaced with a Q Exactive HF (Thermo Fisher Scientific).
Project description:Proteomic Analysis. The proteomic expression of CGMCC 6315 under different nutrient concentration conditions was investigated by isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. The CGMCC 6315 was cultured in LB broth and 1/15LB broth as described above, after which the strains were collected by centrifugation at 10,000× g for 10 min at 4°C. Protein extraction, digestion, iTRAQ labeling and peptide fractionation were performed using the protocol described by Jin et al. 29. Protein identification was conducted using a LC-20AD nano-HPLC instrument (Shimadzu, Kyoto, Japan) equipped with a Q EXACTIVE tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) for data-dependent acquisition detection by nano-electrospray ionization. The raw MS/MS data were converted into MGF format by the thermo scientific tool Proteome Discoverer, and the exported MGF files were searched using Mascot (version 2.3.02) against the selected database containing 7546 CGMCC 6315 coding genes. The IQuant software was used for quantitative analysis of the labeled peptides with isobaric tags. Fold changes of >1.7 with p-values <0.05 were used as a cut off for differentially regulated proteins.
Project description:Female BVSC clone H18 mESCs were cultured in 2i+LIF culture medium (N2B27 Media, CHIR99021 [30 µM; NC9785126; Thermo Fisher], PD0325901 [10 µM; NC9753132; Thermo Fisher], ESGRO Leukemia Inhibitory Factor (LIF) [1000 U/mL, ESG1106; Sigma-Aldrich]) for 72 hr. Differentiation of ESCs to EpiLCs was performed by seeding the cells on Human Plasma Fibronectin (HPF)-coated plates [16.7 µg/mL; 33016015; Thermo Fisher] in the presence of EpiLC induction medium (N2B27 medium containing activin A [20 ng/mL; 50-398-465; Thermo Fisher]), basic fibroblast growth factor (bFGF) [12 ng/mL; 3139FB025; R&D Systems], and KnockOut Serum Replacement [KSR, 1%; Thermo Fisher]. EpiLC samples were collected 48 hours after starting differentiation. Samples were mock treated with water.
Project description:HEK293T cells were ectopically expressing Flag tagged p62 (p62 group) or empty vectors (Control group) for 48h, cell lysates were incubated with anti-Flag affinity gels, and co-immunoprecipitates were subjected to trypsin digestion followed by mass spectrometry analysis. Peptides were separated by the EASY-nLC system (Thermo Fisher) and analyzed by the Q Exactive mass spectrometer (Thermo Fisher). Protein analysis were performed with Thermo Proteome Discoverer 2.1 (Thermo Fisher) and searched against Uniprot Human database. Three samples per group.
Project description:HEK293T cells were ectopically expressing Flag tagged p62 (p62 group) or empty vectors (Control group) for 48h, cell lysates were incubated with anti-Flag affinity gels, and co-immunoprecipitates were subjected to trypsin digestion followed by mass spectrometry analysis. Peptides were separated by the EASY-nLC system (Thermo Fisher) and analyzed by the Q Exactive mass spectrometer (Thermo Fisher). Protein analysis were performed with Thermo Proteome Discoverer 2.1 (Thermo Fisher) and searched against Uniprot Human database. Three samples per group.
Project description:To explore the cellular pathways and the molecular functions affected by a novel biogenic amine, (3-HKA), we performed a label free quantitative (LFQ) proteomic analysis of mouse lymphatic endothelial cells (LEC), and primary dendritic cells, treated with IFNgammaplus or minus 3-HKA. Biological triplicates, of total cell lysates, were fractionated by one-dimensional gel electrophoresis (1DEF) and the “in gel” tryptic derived peptides analyzed by nano-LC-ESI-MS/MS on a Q Exactive HF quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). LFQ analysis highlighted that 3-HKA downregulated many of the inflammatory pathways, more notably JAK/STAT1 and NFkB, associated with IFNgamma activation.
Project description:<p>This dataset comprises untargeted LC-MS metabolomics profiles of rumen samples from 8 sheep under moderate grazing with supplementary feeding and 8 sheep under intensive grazing with supplementary feeding. Samples were analyzed using Thermo Fisher Scientific mass spectrometers in positive/negative ionization modes. Data were processed with Progenesis QI and are intended for identifying differential metabolites in the rumen between the two grazing intensities with supplementation.</p>