K. Nokihara et al., 2-DIMENSIONAL ELECTROPHORESIS AS A COMPLEMENTARY METHOD OF ISOLATING PEPTIDE-FRAGMENTS OF CLEAVED PROTEINS FOR INTERNAL SEQUENCING, Journal of chromatography, 676(1), 1994, pp. 233-238
To determine the primary structure of proteins, usually proteolytic en
zyme digests are separated by reversed-phase high-performance liquid c
hromatography (HPLC) and each fraction is collected and sequenced. The
results obtained by different cleavages are combined to reveal the en
tire 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 successful
ly isolated by the use of two-dimensional electrophoresis, after diges
tion. Electroblotted spots were easily sequenced to identify those dif
ficult fragments which could not be obtained using HPLC.