Tmt kit

Protein extraction was carried out according to a previously described method^{54 (link)}, and the final protein concentration was quantified using the BCA (Beyotime Biotechnology, Beijing, China) assay as per the manufacturer’s guidelines. The proteins’ disulfide bonds of the samples were broken by mixing with a solution of 10 mM DTT for 1 h. Thereafter, these samples were incubated in 50 mM iodoacetamide for alkylation at room temperature for 1 h away from light. Trypsin was used for protein digestion at a volume ratio of 1:50 at 37 °C for 14 h and 1 μL of formic acid was added to the solution for stopping the enzymatic digestion. Finally, peptides were concentrated using a SpeedVac system (Marin Christ, Osterod, Germany). The sample peptide was labeled with TMT kits (Thermo Fisher Scientific, USA) as per the manufacturer’s guidelines. The samples were then combined and stored at -80 °C until the following LC/MS analysis. the peptide sample was loaded onto an LC–MS system. The peptide enrichment, eluted, MS/MS data collected and saved according to a previously study^{54 (link)}. Data are available via ProteomeXchange with identifier accession PXD022768.

All raw files were analysed using MaxQuant software (version 1.5.3.30) queried against the Uniprot complete human database (October 2017). The following search parameters were used: Reporter ion MS2 with multiplicity 6plex TMT for the TMTsixplex experiments and 10plex TMT for the TMT10plex experiments, trypsin digestion with maximum 2 missed cleavages, oxidation of methionine and acetylation of protein N-termini as variable modifications, carbamidomethylation of cysteine as a fixed modification, minimum peptide length of 6, protein FDR 0.01. Appropriate correction factors for the individual TMT channels for both peptide N-terminal labelling and lysine side-chain labelling as per the TMT kits used (Thermo Scientific) were configured into the integrated Andromeda search prior to the MaxQuant search.

To identify the differentially expressed proteins between ccRCC and adjacent normal tissues, equal amounts of proteins were extracted from cancers (n=5) and corresponding adjacent normal tissues (n=5), respectively. The protein concentration was measured with BCA kit (Beyotime Biotechnology, China) according to the manufacturer's instructions. The tryptic Peptide was reconstituted in 0.5 M TEAB (Sigma, USA) and labelled according to the manufacturer's protocol for TMT kit (Thermo Fisher Scientific, USA). Then labelled peptides were mixed and fractionated into 9 fractions using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length), which were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75um i.d.). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive TM Plus (Thermo Fisher Scientific, USA) coupled online to the UPLC. 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.