Preparation method of specific peptide fragment mass spectrometry sample

[0023] Embodiment 1: Preparation method of HEK293T cell-specific peptide mass spectrometry analysis sample:

[0024] A: Take the HEK293T cells to be analyzed, collect 5 million cells in a biotin culture environment, and dissolve them in 2 ml of cell lysis buffer. The composition of the cell lysis buffer is 50 mM triethylammonium bicarbonate, 8 M urea, 10nm protease inhibitor, lysed by circulating ultrasound in an ice bath, quantified the concentration of the protein solution, and then digested the peptide with trypsin;

[0025] B: Trypsin method to digest peptides, a total of 15 mg protein lysis solution, add dithiothreitol at a final concentration of 5 mM, incubate at room temperature for 1 hour, then add iodoacetamide at a final concentration of 10 mM, and incubate at room temperature in the dark For 20 minutes, finally add 3 times the total volume of 50 mM triethylammonium bicarbonate and 150 µg trypsin, and incubate at 37°C with shaking for more than 12 hours; the digested...

[0033] Example 2: HEK293T cell example of Myc-biotididase-KRAS fusion protein

[0034] (1) Preparation of HEK293T cells expressing Myc-biotidase-KRAS fusion protein

[0035] The gene-synthesized fusion gene fragment containing Myc-tag biotinidase (5' end) and KRAS (3' end) was inserted into the pBABE plasmid behind the SV40 promoter through homologous recombination ( figure 2). The fusion gene fragment plasmid was transiently transfected into HEK293T cells using lipofectamine3000 and packaged into retrovirus. HEK293T cells were infected with virus that was filtered through a 0.22 μm filter to remove cell debris for 24 hours, and pressure-selected with 1 μM puromycin for 1 week, and single clones were selected and transferred to 96-well plates for further culture for 2 weeks. image 3 As shown, the specific Myc monoclonal antibody was used to identify the Myc-biotididase-KRAS target band (55 kDa in size) in Western blotting, and cell clones highly expressing the fusion prote...