A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides.
We describe a simple and inexpensive method for determining the location of phosphoamino acids in an isolated phosphopeptide. Phosphopeptides are immobilized on arylamine membrane discs using water-soluble carbodiimide, and the immobilized peptides are subjected to manual Edman degradation. The use of a membrane disc resulted in excellent recovery (65 to 80%) of radiolabeled phosphate following Edman degradation. Furthermore, interference from carryover and peptide washout is reduced to a minimum. The methodology should therefore be able to generate reliable results when used to analyze phosphopeptides that contain multiple phosphorylation sites. In addition to its advantage in sensitivity, the manual sequencing method is easy to perform and does not entail any sophisticated instrumentation.
Sullivan
S
S
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Wong
T W
TW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000737:chemistry
D000596:Amino Acids; D010748:Phosphopeptides; D012319:RNA Polymerase II