Tandem mass tag tmt

Cells were washed and harvested with ice-cold PBS. The proteins were extracted with phase-transfer surfactant (34 (link)) using a lysis buffer (12 mM sodium deoxycholate [Fujifilm Wako], 12 mM sodium N-lauroylsarcosinate [Fujifilm Wako], 100 mM Tris-HCl [pH 9.0], containing protein phosphatase inhibitor cocktail 1 and 2 [Sigma-Aldrich], and protease inhibitors [Sigma-Aldrich]). Protein amount was determined with a BCA protein assay kit, and the proteins were reduced with 10 mM DTT for 30 min and then alkylated with 50 mM iodoacetamide for 30 min in the dark. After reduction and alkylation, proteins were digested with Lys-C (w/w 1:100) for 3 h, followed by trypsin digestion (w/w 1:100) overnight at 37 °C. Then, the peptides were desalted using SDB-XC StageTip.
Phosphopeptides were enriched from 100 μg of tryptic peptides by means of TiO₂-based hydroxy-acid-modified metal oxide chromatography (HAMMOC) (35 (link)) and eluted with 0.5% piperidine. Phosphopeptides were labeled with tandem mass tag (TMT) (Thermo Fisher Scientific), desalted using SDB-XC StageTips, fractionated at basic pH (33 (link)), and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent LC/MS/MS analyses.

Proteins were extracted from kidney tissues by 8 M urea containing 1% protease inhibitor mix. Protein (200 μg) disulfides were reduced by 5 mM Tris (2-carboxyethyl) phosphine (TCEP) for 10 min at room temperature. Then, free cysteine residues were alkylated by 10 mM iodoacetamide for 30 min in the dark. After dilution with PBS, proteins were digested with sequencing-grade trypsin for 16 h at 37 °C at a substrate/enzyme ratio of 50:1 (w:w). Peptides were desalted using Sep-Pak C18 Cartridge (Waters, 186004619), vacuum dried, and labeled with Tandem Mass Tag (TMT, Thermo, Waltham, MA). Mixed peptides were fractionated using the high-pH reversed phase chromatography followed by nano LC-MS/MS analysis.
The MS/MS spectra were searched against the UniProt mus musculus database (June 24, 2017; 59,594 sequences) processed by the Sequest HT search engine using Proteome Discoverer (PD) 2.1 software. The following parameters were used for searching: fixed modifications of carbamidomethylation on cysteine and TMT 6-plex on lysine and peptide N-terminus; variable modification of oxidation on methionine; two trypsin missed cleavages were permitted; the tolerances of precursor and fragment mass were 10 ppm and 0.02 Da; at least two unique peptides for identification of proteins; and 1% FDR (false discovery rate) tolerance at peptide-spectrum match (PSM) level.

Protein extraction from cell pellets, trypsin digestion and enrichment of phosphopeptides were performed as described previously^{55 (link)}. Enriched phosphopeptides for each sample were labelled with Tandem Mass Tag (TMT, Thermo Fischer) according to the manufacturer’s protocol at one-tenth scale.
TMT-labelled samples were combined in each set of experiments and then acidified with trifluoroacetic acid. The combined sample was desalted and trapped by octadecyl (C18)-strong cation exchange using StageTip^{56 (link), 57 (link)}, then fractionated into 7 fractions by elution with each of the buffers as described previously^{55 (link)}. These 7 fractions of TMT-labelled samples were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS).