Micro-identification of amino-terminal acetylamino acids in proteins

By using a reversed phase HPLC system, we have developed a rapid and quantitative micro-identification method for amino-terminal acetylamino acids in proteins at the sample range of 10--100 nmol. The process of the identification method consists of the following steps: (1) digestion of a protein by an appropriate protease; (2) separation of an acidic peptide(s) from the digest on a Dowex 50 x 2 column; (3) further purification of the acidic peptide(s) by reversed phase HPLC using a C18 column; (4) subsequent exhaustive digestion of each acidic peptide to amino acids and an acylamino acid, by pronase and carboxypeptidase Y; (5) isolation of the acylamino acid on a Dowex 50 x 2 column; (6) identification of the acylamino acid on a C18 column; (7) confirmation of the acyl group by HPLC as an 1-acyl-2-dansylhydrazine derivative and of the amino acid on amino acid analyzer. By applying this new method to such proteins as ovalbumin and cytochrome c, their amino-terminal acetylamino acids can be determined in the range of less than 10 nmol.