Triple Protease Digestion PTMs

Triple Digestion Meng-Qiu Dong 6/1/04

1. dissolve proteins in 8 M urea, 100 mM Tris pH 8.5 (60 ul).

2. add 0.3 ul 1 M TCEP (5 mM final conc.). Incubate at RT for 20 min.

3. add 1.2 ul 500 mM iodoacetamide (10 mM final conc.) (make fresh daily, 0.046 g/500 ul H2O). incubate at RT for 15 min in the dark.

4. divide into three equal parts. 5. Trypsin Digest

1. Dilute sample by a factor of 4 (i.e. 60 ul + 20 ul) with 100 mM Tris pH 8.5 (final conc. = 2 M)

2. add 100 mM CaCl2 to a final conc. of 1 mM (i.e. 0.8 ul/80 ul) to inhibit chymotrysion activity

3. add in trypsin 1 ul (0.5 ug/ul)

4. incubate at 37 ºC O/N in the dark.

6. Elastase Digest

5. Dilute sample by a factor of 4 (i.e. 60 ul + 20 ul) with 100 mM Tris pH 8.5 (final conc. = 2 M)

6. add in elastase 1 ul (0.5 ug/ul)

7. incubate at 37 ºC O/N in the dark.

7. Subtilisin Digest

8. dilute with 1/4 vol. of 100 mM Tris pH 8.5 to bring urea to 6M (i.e. 5ul +20 ul) 9. add in subtilysin 1 ul (0.5 ug/ul)

10. incubate at 37 ºC for 4 hr in the dark.

8. Recombine 3 fractions into 1 sample.

9. Add 18.5 ul 90% formic acid (4~5% final conc.).