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.