Protocol (for complex samples)

1. Sample: If sample is already in solution, go to step 2. If it is TCA or acetone precipitation sample, resuspend pellet in 50 mM di-Ammonium Phosphate or 50 mM Ammonium Biocarbonate.
2. Check the pH by spotting ½ µL onto pH paper. Add NH4HCO3 to 50 mM to bring the pH to approximately 8 and 10% ACN.
3. Add denaturant to 0.1% SDS from stock solution (2 to 10% SDS) or 6M Guanidine.
4. Add Tris(2-carboxyethyl) phosphine (TCEP) to 10 mM final concentration and heat at 60C for 45 minutes and allow to cool to room temp.
5. Add freshly made iodoacetamide to 30 mM final concentration and incubate for 1 hour at room temperature preferably in a dark drawer.
6. Add DTT to 30 mM to quench the iodacetamide and wait ten minutes.
7. Add CaCl2 to final 1 mM.
8. Add freshly diluted trypsin into above sample at a ratio of 1:30.
9. Incubate for 4 hours to overnight at 37 C.
10. Spin down the digest and add additional freshly made trypsin at 1:60.
11. Incubate for additional 4 hours at 37 C.
12. Check digest by MS to make sure digestion is completed.
13. Add and equal volume of 1% TFA.
14. Run SPE (Sep-pack) for desalting and elute all tryptic peptides in 90% ACN-0.2% Formic acid.
15. Speed Vac the eluent to less than 50 µL for the subsequent SCX analysis.

Stock solution

• SDS: 2%
• TCEP: 500 mM
• IDA: 200 mM
• DTT: 200 mM
• Ambic: 500 mM
• CaCl2: 500 mM