Metabolomic profiling of primary mouse proximal tubule cells (mPTCs) derived from 24-week-old CTR and Atg7 PT-KO kidneys

ABSTRACT: To explore the role of ATG7 in the kidney PT, we crossed mice homozygous for floxed alleles of Atg7 with Ggt1-Cre mice expressing Cre recombinase under the rat Ggt1 (gamma-glutamyltransferase 1) gene promoter. This generated Atg7fl/fl; γGt1cre/cre mice (hereafter referred to as Atg7 PT-KO), in which exon 14 of Atg7 was efficiently excised, and control littermates (Atg7fl/fl; γGt1wt/wt, referred to as CTR). Sample Preparation. Cellular metabolism was quenched by adding 80% pre-cooled methanol to the cells. After vortexing, samples were incubated at –20°C for 1.5 h. Proteins were precipitated by centrifuging at 10,000 rpm for 20 min at 4°C, and the clear supernatant was transferred to a new vial. The supernatant was then dried under a nitrogen stream and reconstituted in 20 μL water mixed with 80 μL injection buffer (90% acetonitrile, 8.8% methanol, 50 mM ammonium acetate, pH 7). Following another round of vortexing and centrifugation (10,000 rpm, 4°C, 10 min), the clear supernatant was transferred to Total Recovery Vials (Waters) for LC-MS injection. Method blanks, standard mixes, and pooled samples were prepared identically for quality control. LC-MS Data Acquisition. Cell metabolites were separated using a Waters nanoAcquity UPLC with a BEH Amide capillary column (150 μm × 50 μm × 1.7 μm). A gradient was applied using solvent A (5 mM ammonium acetate in water) and solvent B (5 mM ammonium acetate in acetonitrile), transitioning from 5% A to 50% A over 12 min. The injection volume was 1 μL, with the flow rate adjusted from 3 to 2 μL/min. The UPLC was coupled to a Waters Synapt G2Si mass spectrometer via a nanoESI source. MS1 and MS2 data were acquired in negative ion mode using MSE over a mass range of 50 to 1200 m/z at resolutions greater than 20,000. Species: Mus musculus Samples list: Supernatant_0529_WT_Fed Supernatant_0530_WT_Fed Supernatant_0221_WT_Fed Supernatant_0222_WT_Fed Supernatant_0491_cKO_Fed Supernatant_0435_cKO_Fed Supernatant_0436_cKO_Fed Supernatant_0438_cKO_Fed

INSTRUMENT(S): Liquid Chromatography MS - negative - capillary-hilic-/-beh-amide

PROVIDER: MTBLS15857 | MetaboLights | 2026-09-30

REPOSITORIES: MetaboLights