Reprosil particles

Samples were loaded onto 50-cm columns packed in-house with C18 1.9 μM ReproSil particles (Dr. Maisch GmbH), with an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to the MS (Q Exactive HFX; Thermo Fisher Scientific). A homemade column oven maintained the column temperature at 60°C. Phosphopeptides were eluted with a 140 min gradient starting at 5% buffer B (80% ACN, 0.1% Formic acid) followed by a stepwise increase to 20% in 85 min, 40% in 35 min, 65% in 10 min and 80% in 2 × 5 min at a flow rate of 300 nl/min. Samples were measured in data-dependent acquisition with a (TopN) MS method in which one full scan (300–1,650 m/z, R = 60,000 at 200 m/z, maximum injection time 120 ms) at a target of 3 × 10⁶ ions was first performed, followed by 10 data-dependent MS/MS scans with higher energy collisional dissociation (AGC target 10⁵ ions, maximum injection time at 120 ms, isolation window 1.6 m/z, normalized collision energy 27%, R = 15,000 at 200 m/z). Dynamic exclusion of 40 s and the Apex trigger from 4 to 7 s was enabled.

Each mouse kidney tissue was ground into powder in nitrogen., then lysis buffer (0.1 M Tris-HCL, pH 7.5, 4% SDS, 0.1 M DTT) was added, and the mixture was sonicated with 20 cycles of pulses (30 s each on/off, 80% power; CosmoSonic II Ultra Sonicator). After heating for 10 min at 95 °C, the lysate was centrifuged at 16,000 × g for 20 min at room temperature. The protein concentrations were determined by measuring tryptophan fluorescence^{66 (link)}. 100 μg protein was digested by the FASP method^{67 (link)}. Each sample peptides were loaded onto a 20-cm column packed in-house with C₁₈ 3 μM ReproSil particles (Dr. Maisch GmbH), with an EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to the mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific). Column temperature was maintained at 50 °C. Peptides were separated with a 120 min gradient at a flow rate of 300 nL/min. Each sample was detected twice. The raw data files were processed using software MaxQuant (http://www.maxquant.org.) version 1.6.2.10 with an FDR < 0.01 at the levels of proteins and peptides. The MS/MS spectra were searched against the Homo sapiens protein database in UniProt (January 2021). Bioinformatics analyses were carried out with R version 4.1.0 (https://www.r-project.org/) statistical computing software.