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).