Notes:
1.
Please always wear gloves and a lab coat to avoid keratin contamination.
2.
Please use clean but not autoclaved tips and microcentrifuge tubes.
Reagents
( Use HPLC grade solvents, highest possible grade reagents and MilliQ water for all preparations)

50 mM Tris, pH 8.0

8 M Urea/50 mM Tris, pH 8.0

Reducing reagent ： 200 mM DTT/50 mM Tris, pH 8.0

Alkylation reagent ： 200 mM Iodoacetamide/50 mM Tris, pH 8.0

Trypsin solution (2μg/μl): keep on ice before use.
Procedure:
1. Reconstitute the protein pellet (0.1-1 mg) in 30μl of 8 M Urea/50 mM Tris, pH 8.0.
(protein concentration ： 10-30 μg/μl)
2. Add 2μl of 200 mM DTT/50 mM Tris, pH 8.0, and incubate the mixture for 2.5 h at room temperature.
3. Add 6μl of 200 mM Iodoacetamide/ 50 mM Tris, pH 8.0, gentle vortex, and incubate the mixture for 1 h at room temperature in dark.
4. Add 6μl of 200 mM DTT/ 50 mM Tris, pH 8.0, and incubate the mixture for 1 h at room temperature in dark.
5. Add 250μl of 50 mM Tris to reduce the concentration of Urea.
6. Add trypsin solution in a ratio of 1:50 (w/w, trypsin : protein. For example: 10μl of
2μg/μl trypsin solution for 1mg protein). Gentle vortex and incubate at 37
℃ overnight.
7. Peptide digests are further purified by C18 Zip-tip, which can be purchased from core lab. For detail procedures, please refer to Zip-tip user's manual.
(Please note that, to eliminate the damage of UPLC column, step 7 is a "must-do” procedure before your submission.)
8. Purified peptide digests are lyophilized, and then store at -20C