1. Resuspend pellet in 6M GnHCl 100 mM tris pH 8
2. Boil 5 min
3. Rest 5 min
4. Boil 5 min
5. QS with MeOH to 90% [MeOH]
6. Centrifuge 9,000x g for 5 min
7. Decant (remove excess supernatant with pipette)
8. Air dry 5 min
9. Resuspend in 8M urea & 100 mM Tris (should be <3 mg/ml)
   • Probe sonicate until resuspended
10. Quantify protein by BCA assay (or method of choice)
11. Add 10 mM TCEP, 40 mM chloroacetamide
12. Add LysC 50:1, incubate 4 hrs
13. QS with 100 mM tris to 2 M urea
14. Add trypsin 50:1, incubate overnight
15. Add TFA to pH ~1
16. Centrifuge 9,000x g for 5 min

Desalting (≤300 µg)

10 mg Strata X, 1 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

1. 1 mL 100% ACN
2. Fill cartridge with 0.2% FA
3. Load sample
4. Fill cartridge with 0.2 % FA
5. Elute into clean tube with 300 µl 80% ACN 0.2% TFA.
6. Dry down reconstitute in 0.2% FA.
   

Desalting (≤1 mg)

30 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

1. 3 mL 100% ACN
2. Fill cartridge with 0.2% FA
3. Load sample
4. Fill cartridge with 0.2 % FA
5. Elute into clean tube with 900 µl 80% ACN 0.2% TFA.
6. Dry down reconstitute in 0.2% FA.

Desalting (≤5 mg)

60 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

1. 3 mL 100% ACN
2. Fill cartridge with 0.2% FA
3. Load sample
4. Fill cartridge with 0.2 % FA
5. Elute into clean tube with 1.5 mL 80% ACN 0.2% TFA.
6. Dry down reconstitute in 0.2% FA.

Desalting (≤10 mg)

100 mg Strata X, 3 mL volume (reversed phase solid phase extraction cartridge)

Note: Use gravity or a vacuum manifold

1. 3 mL 100% ACN
2. Fill cartridge with 0.2% FA
3. Load sample
4. Fill cartridge with 0.2 % FA
5. Elute into tube with 2 ml 80% ACN 0.2% TFA.
6. Dry down reconstitute in 0.2% FA.

Peptide concentration

Resuspend sample in 0.2% FA, targeting ~2 µg/µL

Use Peptide colorimetric assay for peptide quantitation.