Easy nlc 1200 chromatographic system

The specific antibodies were incubated with Protein A/G magnetic beads (bimake) at room temperature for 4 h. Then, the extracted proteins were added into the mixtures of antibodies and beads at room temperature for 1 h. The bead-bound proteins were subjected to western blotting or MS.
For MS, the bead-bound proteins were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Then the gels were digested, and the peptides were obtained from the gels. The anti-p66Shc IP-specific peptides were separated by an Easy nLC 1200 chromatographic system (Thermo Scientific) and then analyzed on a Q-Exactive HF-X mass spectrometer (Thermo Scientific). MaxQuant 1.6.1.0 was used to analyze the LC-MS/MS data. Peptide-spectrum match (PSM) false discovery rate (FDR) ≤ 0.01 and Protein FDR ≤ 0.01 were used to identify the peptide and protein, respectively.

Cells were cultured to > 90% confluence then lysed in immunoprecipitation lysis buffer (Beyotime Shanghai, China) with protease and protein phosphatase inhibitors. Lysates were incubated with 20 μL of protein A/G magnetic beads (MedChemExpress, New Jersey, USA) for 2 h. After incubating, the beads were removed, and 5–10 μL of primary antibody (KSR2) or isotype IgG was added to the supernatant and the samples mixed with gentle rocking at 4 °C overnight to capture the fusion proteins. Then, 20 μL of protein A/G beads was added and the immunoprecipitation mixes incubated for 2 h. Magnetic beads were collected by placing the tube in the appropriate magnetic separator. Beads were washed three times with cooled PBS buffer to remove nonspecifically bound proteins. Bound fusion proteins were eluted from the beads. Peptides were then analyzed (Bioprofile, Shanghai, China) with the Easy-nLC1200 chromatographic system (Thermo Fisher, Massachusetts, USA) and Q-exactive Plus mass spectrometer (Thermo Fisher, Massachusetts, USA). Protein identification was performed using MaxQuant1.6.1.0.

The label-free quantitative proteomics method was used for MS by Shanghai Bioprofile Technology Co., Ltd. Cell pellets were harvested, and total protein was extracted using SDT cell lysis reagent. Digested peptides were desalted using peptide desalting spin columns and lyophilized under vacuum. Peptide concentrations were measured using a Nanodrop. When performing the MS for phosphorylated proteins, the peptide solution was lyophilized under vacuum, and phosphorylated peptides were enriched with an Fe-NTA Phosphopeptide Enrichment Kit (Thermo, A32992); enriched phosphorylated peptides were collected according to the kit procedure for mass spectrometry analysis. An appropriate amount of enriched peptides for each sample was separated using a nanoliter flow rate Easy nLC 1200 chromatographic system (Thermo Scientific). MSFragger 3.4 software was used to retrieve data from UniProt Protein Data Bank (UniProt Homo sapiens (Human) [9606]-203800-202201.fasta). After comparison, a log2 (fold change) ≥ 1.5 (total proteins) or 2 (phosphorylated proteins) and P ≤ 0.05 were considered to indicate significantly different expression of modifier sites.