Determination of the relative positions of amino acids by partial specific cleavages of end-labeled proteins.
We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007798:Lampreys; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins; D013045:Species Specificity; D013861:Thiocyanates; D014357:Trypsin
D000596:Amino Acids; D003619:Dansyl Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D013861:Thiocyanates; D005340:Fibrinogen; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate