Studies on the methods for the determination of phosphorylation sites in highly phosphorylated peptides or proteins: phosphorylation sites of hen egg white riboflavin binding protein.
To determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the beta-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and beta-methylaminoalanines (DL-alpha-amino-beta-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for determining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: His182-Leu-Leu-Ser185-Glu-Ser(P)-Ser(P)-Glu-Glu190-Ser (P)-Ser(P)-Ser(P)-Met-Ser195(P)-Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phosphoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D002645:Chickens; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism; D005260:Female; D007700:Kinetics; D026901:Membrane Transport Proteins; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D004527:Egg Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D010710:Phosphates; C019079:riboflavin-binding protein; D010768:Phosphoserine; C008425:S-methylcysteine; D003545:Cysteine