Proteomics
Protein was sequenced by PTM-Biolab Co. Ltd (Shanghai, China). Kidney tissue containing 30 mg of protein was digested with trypsin. After digestion, eluates were desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. TMT labeling was performed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. The tryptic peptides were fractionated by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Samples were dried down and were stored at -20°C before LC-MS/MS measurement.
Fractions (0.1% formic acid) were subjected to NSI source followed by MS/MS in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
MS/MS data were identified by matching the raw data to the UniProtKB mouse database (version v06.06.14) using MaxQuant version 1.5.2.8 and its built-in Andromeda search engine for peak detection and quantification. Search parameters were set as follows: (i) full tryptic specificity was up to 4 missing cleavages; (ii) mass tolerance for precursor ions was 20 ppm in First search and 5 ppm in Main search; (iii) mass tolerance for fragment ions was 0.02 Da; (iv) carbamidomethyl on cysteine was set as fixed modification; and (v) acetylation modification and oxidation on Met were specified as variable modifications. FDR was adjusted to < 1% and minimum score for modified peptides was set > 40.