The Determination of Amino Acid Sequence Abnormalities in Proteins by HPLC Peptide Analysis

Current techniques for DNA amplification and sequencing have greatly simplified the identification of genetic mutations underlying disorders of abnormal protein production. The need, however, for the direct characterization of an amino acid abnormality in a defective protein still arises in particular situations. The normal approach to this problem relies on the specific fragmentation of the protein and subsequent analysis of the resultant mixture of peptides. The techniques currently available for peptide analysis offer very high sensitivity and so require relatively small amounts of protein. Automated peptide sequencing or mass spectrometry can be achieved with subnanomole quantities of peptide. Consequently, sufficient material can be isolated from tryptic digests beginning with as little as a milligram or so of a pure protein.