General method for the separation of cyanogen bromide digests of proteins by high-performance liquid chromatography....

In this study, we have demonstrated the necessity for a combination of size-exclusion, ion-exchange and reversed-phase high-performance liquid-chromatography to resolve completely a protein digest. Our approach minimizes the number of steps, and the column order provides the maximum information about the properties of the fragments. The order is: (1) size-exclusion (Bio-Rad TSK-250 column), (2) strong cation-exchange (Synchropak S300 column), and finally (3) reversed-phase chromatography (Ultrapore C3). It was desirable for the first step of the procedure to be size-exclusion chromatography to produce the least number of fractions. The volatile eluent used in size-exclusion eliminated the need for subsequent sample desalting. Volatile buffers were not necessary for the ion-exchange chromatography, since the fractions were both desalted and purified in the final reversed-phase step. All column effluents were compatible with absorbance measurements at 210 nm to provide maximum sensitivity for peptide detection. The results obtained in this study strongly suggest that the combined use of three methods of separation, which utilize different selectivities (size, charge, hydrophobicity), can provide excellent resolving power for peptide separations. We believe this fast, efficient procedure should be generally applicable to other protein digests.