- Quantitative Proteome 29
- 200 mM S-Methyl methanethiosulfonate (MMTS) (500μL)
Mix 9. 73 μL of MMTS and 490. 27 μL of Milli-Q water. Store at 4◦Cand
avoid exposure to light. - 10% Ammonium persulfate (APS) (1 mL)
Dissolve 0.1 g of APS powder in 1 mL of Milli-Q water. Store at− 20 ◦C. - 25 mM Triethylammonium bicarbonate buffer (TEAB) (50 mL)
Mix 1.25 mL of 1 M TEABC and 48.75 mL of Milli-Q water. Store it at
room temperature. - 25 mM TEAB/50% Acetonitrile (ACN) (50 mL)
Mix 1.25 mL of 1 M TEABC, 25 mL of 100% ACN and 23.75 mL of Milli-Q
water. Store it at room temperature. - 10% Trifluoroacetic acid (TFA) (10 mL)
Mix 1 mL of 100% TFA and 9 mL of Milli-Q water. Store at room
temperature. - 0.1% TFA (50 mL)
Mix 0.5 mL of 10% TFA and 49.5 mL of Milli-Q water. Store it at room
temperature. - 50% ACN/0.1% TFA (50 mL)
Mix 25 mL of 100% ACN, 0.5 mL of 10% TFA and 24.5 mL of Milli-Q
water. Store it at room temperature.
Procedure
Protein extraction from tissue samples
- Grind the tissue into powder with mortar and pestle in liquid nitrogen.
Add liquid nitrogen frequently to ensure that the tissue does not thaw
during grinding. Distribute the powder to 1.5 mL centrifuge tubes. For
long-term storage, this tissue sample can be stored at− 80 ◦C. - Precool the centrifuge and rotor to 4◦C before starting lysis. Prepare
the lysis buffer with volume of about 3–5 times of the volume of tissue
sample.- Be sure to add protease inhibitor before using the lysis buffer.
- Add lysis buffer to tissue sample and pipetting or vortex to mix
thoroughly. - Homogenize the sample solution on ice using homogenizer with 60%
amplitude, cycle of 0.6 (operated 0.6 second every second) for 4–5 minutes
several times until the solution become almost transparent and not
viscous.