Proteomics grade

Protein samples were prepared for mass spectrometry measurements as previously described (29 (link)). In short, the indicated protein complex was subjected to SDS-PAGE. After the sample became a compact slit-like band after loading, the protein band was excised from a Coomassie-stained SDS-PAGE gel and fragmented into small pieces. Thereafter, the proteins were reduced with 10 mM dithiothreitol (DTT) for 30 min at 55°C, followed by alkylation with 55 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. Overnight digestion at 37°C was performed with 0.6 μg of trypsin (Proteomics-grade, Promega) in 25 mM NH₄HCO₃. The resulting peptides were acidified with 0.1% formic acid (FA) to stop trypsin digestion (30 (link)).

Protein samples were prepared for mass spectrometry measurements as previously described (62 (link)). Briefly, the indicated protein complex was subjected to SDS–PAGE. When the electrophoresis was carried out, the sample became a compact slit-like band after loading, the protein band was excised from a Coomassie-stained SDS–PAGE gel and fragmented into small pieces. Thereafter, the proteins were reduced using 10 mM dithiothreitol for 30 min at 55 °C, followed by alkylation with 55 mM iodoacetamide for 30 min at room temperature in the dark. Overnight digestion at 37 °C was performed with 0.6 μg of trypsin (Proteomics-grade, Promega) in 25 mM NH₄HCO₃. The resulting peptides were acidified using 0.1% formic acid (FA) to stop trypsin digestion (63 (link)). After the desalting procedure, the peptides were analyzed by the LTQ Orbitrap Elite system and Easy-nLC 1000 system (Thermo Fisher Scientific).