2.12 Liquid Chromatography-Mass Spectrometry (LC‐MS) analysis
Proteins purified from RNA pull-down assay were analyzed using LC‐MS by the Shanghai Applied Protein Technology Co., Ltd (Shanghai, China). Two libraries (CRIR1 and Anti-CRIR1 ), which contained two replicates for each library, were prepared in this study. Dry pellets of samples were solubilized in trypsin buffer (Promega) for digestion into small peptides. The digested peptides were desalted and solubilized in 0.1% formic acid buffer. The peptide mixture was loaded onto a reverse-phase trap column (Thermo Scientific Acclaim PepMap100) connected to the C18 reversed-phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid). LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific). MS data were acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300-1800 m/z) for HCD fragmentation. Data were analyzed for each sample with MaxQuant software (Cox & Mann, 2008).