Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing.

To determine the primary structure of proteins, usually proteolytic enzyme digests are separated by reversed-phase high-performance liquid chromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the entire sequence. However, there are many N-terminal-blocked proteins and/or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successfully isolated by the use of two-dimensional electrophoresis, after digestion. Electroblotted spots were easily sequenced to identify those difficult fragments which could not be obtained using HPLC.