The insoluble residue was separated by centrifugation (1,520g for 20 min at 4°C), and the supernatant was dialyzed against 0.2 M ammonium acetate (pH 7.0) at 4°C overnight. Dialysis was repeated twice, against 0.02 M and then 2 mM ammonium acetate. The samples were concentrated by using a Vivaspin 20 (MWCO 10,000; Vivascience) and transferred to a HiTrap desalting column (Amersham Biosciences).