Materials:

Note: Make 8M Urea in Tris and IAA fresh!

8M Urea in 100 mM Tris-HCl, pH 8.5 Dissolve 480 mg of Urea in 700 ul of 100 mM Tris-HCl (pH 8.5). Make Fresh.

1M TCEP (reducing agent) Dissolve 287 mg TCEP in 1ml  DI water. Aliquot (10 ul) and store

500 mM Iodoacetamide (IAA) (alkylating agent) Dissolve 92 mg in 1 ml DI water.

1 ug/ul Trypsin solution 5 ul aliquots of 1 ug/ul Trypsin in 0.5 ml microtubes are stored in the -80C. (To make the 1 ug/ul stock, mix 100 ul DI  water with 100 ug of Trypsin)

Digestion Procedure

1) Reconstitute dried protein sample in 40 ul of 8.0 M urea in 100 mM Tris-HCl (pH 8.5)

2) Dilute the 1M TCEP 1:10 in DI water and add 1.2 ul of 100 mM TCEP (5 mM final concentration)

and shake for 20 minutes at room temperature.

3) Add 0.88 ul of 500 mM IAA (10 mM final concentration) and shake in the dark for 15 minutes.

4) Add 120 ul of 100 mM Tris-HCl (pH 8.5)

solution to dilute the urea to 2M.

5) Add Trypsin in appropriate ratio (1:30) to approximate amount of protein by weight. (Generally speaking, for a complex sample, such as a whole cell or whole

organelle lysate, a good starting point is 0.5 ug.)

6) Digest overnight at 37oC.

7) Follow C18 spin column protocol to clean up sample (remove urea) before running on mass spec