Computational protocol: CD47 surface stability is sensitive to actin disruption prior to inclusion within the band 3 macrocomplex

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[…] Proteomic analysis was conducted using Nano LC-MS/MS mass spectrometry by the University of Bristol Proteomics Facility. Total CD47 was immunoprecipitated using BRIC126 up to 15 million K562 cells, expanding (EXP) and differentiating erythroblasts (T0 and T48), and mature erythrocytes (n = 2). Briefly, protein G beads were fractionated by 1D SDS-PAGE until the dye front had moved approximately 2 cm into the separating gel. The gel lanes were cut into 2 equal portions and in-gel digested with trypsin. Extracted peptides were subjected to Nano LC-MS/MS mass spectrometry. The raw data files were processed using Proteome Discoverer software v1.4 (Thermo Scientific) and searched against the UniProt Human database (126385 entries) using the SEQUEST (Ver. 28 Rev. 13) algorithm. Peptide precursor mass tolerance was set at 10ppm, and MS/MS tolerance was set at 0.8 Da. Search criteria included carbamidomethylation of cysteine (+57.0214) as a fixed modification and oxidation of methionine (+15.9949) as a variable modification. Searches were performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed. The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate (FDR) of 5%. The Proteome Discoverer software generates a reverse “decoy” database from the same protein database used for the search and any peptides passing the initial filtering parameters that were derived from this decoy database are defined as false positive identifications. The minimum cross-correlation factor (Xcorr) filter was readjusted for each individual charge state separately to optimally meet the predetermined target FDR of 5% based on the number of random false positive matches from the reverse decoy database. Thus each data set has its own passing parameters. […]

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