Affinity purification—mass spectrometry (AP-MS) analysis

Samples were prepared as described in the section “Immunoprecipitation”, and separated by SDS-PAGE. The samples were in-gel digested and subjected to LC–MS/MS analysis. The MS/MS spectra from each LC–MS/MS run were searched against the UniProt human database³² (version 10 January 2015, 89,105 sequences) using an in-house Sequest HT algorithm in Proteome Discoverer 2.1 software (Thermo Fisher Scientific Inc.). The search criteria were as follows: full tryptic specificity was required; one missed cleavage was allowed; oxidation (M) was set as dynamic modification; carbamidomethylation (C) was set as fixed modification; precursor ion mass tolerances were set at 10 ppm for all MS acquired in an Orbitrap mass analyzer; and the fragment ion mass tolerance was set at 0.02 Da for all MS2 spectra acquired in the linear ion trap. The searched data were further processed with the Percolator function in Proteome Discoverer to filter with a 1% peptide false discovery rate (FDR). The SAINT algorithm³³ (http://sourceforge.net/projects/saint-apms) was used to evaluate the MS data. Proteins with at least 2 unique peptides, SEQUEST score ≥ 40 in the overexpression sample and SAINT score ≥ 0.85 were considered as candidate interaction proteins. Candidate interactions were visualized using the STRING database (https://string-db.org/)12 with a threshold of 0.7.