A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten

Materials and Reagents

1. Seven-day old protonemata of the moss P. patens
   
2. Liquid nitrogen
   
3. Tris-HCl (pH 7.5)-saturated phenol (AMRESCO, catalog number: K168 )
   
4. Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
   
5. Methanol (Sigma-Aldrich, catalog number: 34860 )
   
6. DTT (Promega Corporation, catalog number: V3155 )
   
7. Acetone (Sigma-Aldrich, catalog number: 650501 )
   
8. Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
   
9. Ammonium bicarbonate (pH 8.5) (Sigma-Aldrich, catalog number: 40867 )
   
10. Trypsin (Promega Corporation, catalog number: V5111 )
11. Sucrose (Sigma-Aldrich, catalog number: S5390 ) 
    
12. Tris (Sigma-Aldrich, catalog number: T1378 )
    
13.  EDTA (Sigma-Aldrich, catalog number: E6635 ) 
    
14. 1,4-dithiothreitol (DTT) (Promega Corporation, catalog number: V3155) 
    
15. Protease inhibitors cocktail (Sigma-Aldrich, catalog number: P9599 )  
    
16. Phosphatase inhibitors cocktail 2 (Sigma-Aldrich, catalog number: P0044 ) 
    
17. NH₄OH (Sigma-Aldrich, catalog number: 320145 )   
    
18. Urea (Sigma-Aldrich, catalog number: U6504 )   
    
19. CHAPS (Sigma-Aldrich, catalog number: C9426 )  
    
20. Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34851 )   
    
21. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )  
    
22. Glutamic acid (Sigma-Aldrich, catalog number: G0355000 )
    
23. Protein extraction buffer (see Recipes)
    
24. Protein resuspension buffer (see Recipes)
    
25. Loading buffer (see Recipes)
    
26. Washing buffer I (see Recipes)
    
27. Washing buffer II (see Recipes)
    
28. Elution buffer (see Recipes)
    

Equipment

1. Porcelain Mortar and Pestle (90 mm)
   
2. Tubes and tube holder (2 ml)
   
3. Titanium dioxide micro-columns (320 µm x 50 mm) (Column Technology, Freemont)
   
4. Centrifuge
   
5. Vortex
   
6. Freeze dryer (Christ, model: Alpha 1-4 LSC )
   

Procedure