42 A Practical Guide to Cancer Systems Biology
VI. Remove cell debris by centrifuge at 16,000×g for 20 minutes, 4◦C.
VII. Transfer the supernatant to a new 1.5 mL tube. The protein extracts
can be stored at− 80 ◦Cuntiluse.
- Protein digestion and desalting
Materials:
- BCA protein assay kit (#23225, Thermo Scientific)
- Dithiothreitol (DTT) (CAS NO. 3483-12-3)
- Iadoacetamide (IAM) (CAs NO. 144-48-9)
- 1M TEAB/ammonium bicarbonate
- Lys-C endopeptidase (#129-02541, WAKO)
- Sequencing grade trypsin (#90305, Thermo Scientific)
- Ethyl acetate (EtAc) (CAS NO. 141-78-6)
- EmporeTMSDB-XC 6 mL extraction disc cartridge (#4340HD, 3M)
- Solution A: 0.1% TFA, 80% ACN
Procedure:
I. Determine the protein concentration using BCA protein assay kit.
II. Transfer suitable amount of proteins to a new tube. Adjust the protein
concentration to 1–1.5 mg/mL using lysis buffer.
III. Prepare 0.5 M DTT in 50 mM TEAB/ammonium bicarbonate (freshly
prepared).
IV. Protein reduction by adding 0.5 M DTT to a final concentration of
10 mM. Rotating for 30 minutes at room temperature.
V. Prepare 1 M IAM in 50 mM TEAB/ammonium bicarbonate (freshly
prepared).
VI. Protein alkylation by adding 1 M IAM to a final concentration of
50 mM. Rotating for 30 minutes at room temperature in dark.
VII. Add Lys-C to obtain an enzyme: protein ratio (mass: mass) of 1:100.
Rotating for 3 hours at room temperature.
VIII. Add trypsin to obtain an enzyme: protein ratio (mass: mass) of 1:50.
Rotating for 16–18 hours at room temperature.
IX. For detergent removal, add equal volume of EtAc to the peptide
sample.
X. Inactivate trypsin by adding 10% TFA to a final concentration
of 0.5%.
XI. Agitate for 2 minutes to completely mix EtAc and sample solution.