Tandem mass tag (TMT) quantitative analyses of mitochondrial proteomes

To profile mitochondrial proteomes under normoxia and hypoxia, mitochondria were isolated by Cell Mitochondria Isolation Kit (Beyotime Biotechnology, C3601) according to the manufacturers' protocol from T47D cells with normoxia or hypoxia treatment for 24 h, which was performed in triplicates. Then, Applied Protein Technology performed quantitative mitochondrial proteomics by TMT labeling. Briefly, SDT buffer (4%SDS, 100 Mm Tris/HCl Ph7.6, 0.1 M DTT) was used for mitochondria lysis, mitochondria extraction is then digested by filter‐aided sample preparation (FASP). 100 μg peptide mixture of each sample was labeled by Tandem Mass Tag (TMT). Subsequently, the resulting peptides went through fractionation and were analyzed by LC–MS/MS. Data were analyzed by Proteome Discoverer (v.1.4, Thermo Scientific) against the human UniProt Reference Proteome database.