Approaches for submicrosequencing

Abstract

From a conventional SDS-acrylamide gel plate, protein (≈1 μg), even after staining by dye, was extracted with 70% formic acid and purified by Biogel P-10 column chromatography in 70% formic acid. The recovery was 60–99% and the protein purified by this method was free from SDS, dyes, glycine, and buffer salts and ready to use for protein composition and sequence studies. The method can avoid the protease digestion of the protein that has often been observed in the electroelution method. For amino acid analysis, protein was hydrolyzed with a gas of a HCL/trifluoroacetic acid/H₂O mixture at 158°C for 22.5 and 45 min to avoid contamination (less than 1 pmole). The method provided sufficient hydrolysis even for hydrophobic protein. It is under process of automation. Carboxyl-terminal analysis was performed with carboxypeptidases A, B, and P in the presence of alcohols and detergents. The effects on the carboxypeptidases (and several proteases) are summarized. These additions increased the solubility of proteins and enhanced the digestion. For N-terminal sequencing, conventional Edman degradation was followed up to the ATZ-derivative and this ATZ-derivative was reacted with primary amines such as ¹²⁵I-histamine or 1-aminopyrene to produce a phenylthiocarbamyl-amino acid-amine derivative with a yield as high as 95%. The conventional procedure of Edman degradation was preserved, but instead of the acid conversion to PTH derivatives, the above coupling procedure was introduced in order to increase the sensitivity to the 1–100f mole range.