MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14165/m_MTBLS14165_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14165/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14165/s_MTBLS14165.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14165/a_MTBLS14165_LC-MS_positive_reverse-phase_metabolite_profiling.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14165<p>T-test was used to compare Cl-free treated cells and control cells. Significantly differential (p&lt;0.05) genes/proteins were used for KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis through an online platform KOBAS (http://kobas.cbi.pku.edu.cn).&nbsp;&nbsp;</p>MetaboLightsPublicLiquid Chromatography MS - positive - reverse phase<p>Tryptic-digested peptide samples were injected into a Vanquish Neo LC system (ThermoFisher, USA) for peptide separation. Peptides were separated via a trap-and-elute workflow via a 20 mm X 75 µm trap column (ThermoFisher, USA) and an Aurura Ultimate XT 25 cm X 75 µm C18 column (Ionopticks, Australia). The separation column was kept at 50 oC with a XT compatible heater controller (Ionopticks, Australia). Solvent A and B were 0.1% formic acid in milli-Q and 0.1% formic acid in acetonitrile respectively. Gradient settings with 300nl/min flow were as follows: from 2% B to 6% B in 0-2 min, then from 6% B to 30% B in 2-77 min, then from 30% B to 90% B in 79-82 min, being held until 87 min, then ramped down from 90% B to 2% B at 87.1 min and being held until 90 min.</p>Direct Role of CFTR in Bones: Cl- Homeostasis Required for Osteocyte Viability.Ye Chun RuanThe Hong Kong Polytechnic UniversityBonemass spectrometry assay<p>Proteins were extracted and purified by EasyPep mini-MS sample prep kit (Thermo scientific, A40006).A vacuum centrifuge was used to dry the proteins, which were then resuspended in 0.1% formic acid.&nbsp;</p>Mus musculus<p>Proteome Discoverer software (Version 2.1) was used for raw data to be aligned with UniprotKB protein database for Mus musculus (UP000000589) with 55,466 entries. The label-free quantitation (LFQ) algorithm was applied to calculate protein abundances as the sum of the peptide group abundances associated with that protein.&nbsp;</p>CFTR knockoutChloride free treatmentsharon.yc.ruan@polyu.edu.hkhttps://www.ebi.ac.uk/metabolights/MTBLS14165<p>MLO-Y4 cells were incubated in the Margo ringers’ solution or a modified Cl- free solution (in mM): Na-gluconate 130, K-gluconate 5, Ca-gluconate 2.5, MgSO4 1, HEPES 20 and glucose 10 (pH=7.4) at 37° for 2 hours before whole-cell proteins were extracted. In another set of experiments, CFTR knockout and wild-type MLO-Y4 cells were collected for analysis after 48 hours of culture following seeding.</p>MetabolomicsMus musculusuntargeted analysisProteomicsMLO-Y4 cellchlorideBoneCFTRVanquish Neo LC systemOrbitrap Fusion Lumos Mass Spectrometerexperimental sampleMus musculusuntargeted analysisProteomicsMLO-Y4 cellchlorideBoneCFTRVanquish Neo LC systemOrbitrap Fusion Lumos Mass Spectrometerexperimental sample<p>For DIA, Eluted peptides were directly introduced into an Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher, USA) and mass spectra data were acquired using a data-independent acquisition (DIA), with an MS1 scan range of 400–1500 m/z. The spray voltage was set at 2 kV with an ion transfer tube temperature of 300 °C. MS1 resolution was set to 60,000, with an AGC target of 4.00E5 and a maximum injection time of 50 ms. DIA MS2 was then acquired with two mass ranges: 400–650 m/z with an isolation window of 10 m/z, and 650–800 m/z with an isolation window of 15 m/z. Both ranges were acquired with an MS2 resolution of 10,000, a 1000% AGC target, 30% HCD collision energy, automatic injection time, and a scan range of 200–2000 m/z.</p><p>For DDA, Eluted peptides were directly introduced into an Orbitrap Fusion Lumos Mass Spectrometer (ThermoFisher, USA) and mass spectra data were acquired using a data-dependent acquisition (DDA) workflow with a scan range of 400–1500 m/z. The spray voltage was set to 2 kV with an ion transfer tube temperature of 300 °C. MS1 resolution was set to 60,000, with an AGC target of 4.00E5 and a maximum injection time of 50 ms. MS2 was then acquired using a 3-sec cycle time and a 40-sec dynamic exclusion period, with an Orbitrap resolution of 15,000, 30% HCD collision energy, an AGC target of 5.00E4, and a maximum injection time of 22 ms.</p>Not applicablefalseProteomic analysis of osteocyte line (MLO-Y4) with knockout of cystic fibrosis transmembrane conductance regulator (CFTR) or chloride deprivation treatment<p>Mass spectrometry–based proteomic analysis of an osteocyte line, MLO-Y4, was conducted to profile global protein expression changes under two experimental settings, 1) CRISPR-Cas9-based knockout of CFTR versus wild-type; 2) Cl- deprivation versus normal extracellular medium.</p>2026-03-272026-03-27MTBLS14165