이전 사이트 이동

Acetyl (K), Acetyl (Protein N-term), Carbamidomethyl (C), Deamidated (N), Deamidated (Q), Oxidation (M), Phospho (S), Phospho (T), Phospho (Y)

Affinity purification (AP-MS)

Thermo Scientific Orbitrap Fusion Lumos Tribrid

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based ZNF184 interactome analysis To identify ZNF184 interactome proteins, HEK293T and REH cells were transfected with S-FLAG-streptavidin binding peptide (SFB)-tagged ZNF184 or control SFB vector plasmid. SFB-ZNF184 and associated proteins were immunoprecipitated from cell lysates using FLAG-M2 affinity gel (Sigma-Aldrich), and the immunoprecipitated proteins were separated by SDS-PAGE. After staining with colloidal Coomassie blue, bands were sliced from the protein gel and in-gel tryptic digestion was performed as described by Shevchenko et al19. Tryptic digests were analyzed using a Thermo Scientific Eazy-nano LC 1200 UHPLC system coupled with an Orbitrap Fusion Lumos mass spectrometer. Peptides were separated on a reversed-phase trap and analytical column and eluted with a gradient of 3–80% acetonitrile over 80 minutes. MS data were acquired in data-dependent mode with a resolution of 120,000 for MS1 scans, selecting precursors with charges 2–7 and intensity >5e3 for fragmentation. Dynamic exclusion was set to 15 s with 10 ppm mass tolerance.

MS/MS data were searched against the UniProt human database using the SEQUEST algorithm via Proteome Discoverer Sorcerer 2.4. Carbamidomethylation of cysteine was a fixed modification, while oxidation (M), N-terminal acetylation, and phosphorylation (S/T/Y) were variable. Results were validated with Scaffold (v5.3.1), accepting peptides with >97% probability and <1% FDR and proteins with >95% probability, <1% FDR, and at least two peptides, using ProteinProphet algorithm20. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 21 partner repository with the dataset identifier PXD (accession number)