Determination of phosphorylation sites in peptides and proteins employing a volatile Edman reagent.
A manual Edman degradation protocol has been developed that allows the identification of phosphorylation sites in 32P-labeled peptides at the subpicomole level. By using both a volatile reagent, trifluoroethyl isothiocyanate, and volatile buffers, extraction steps are rendered unnecessary and cycle times can be reduced to 45 min. The protocol was employed to identify the site of phosphorylation in phosphoserine- and phosphotyrosine-containing peptides.
Fischer
W H
WH
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037, USA. Fischer@sc2.salk.edu
Hoeger
C A
CA
Meisenhelder
J
J
Hunter
T
T
Craig
A G
AG
D001665:Binding Sites; D002855:Chromatography, Thin Layer; D004586:Electrophoresis; D006801:Humans; D017879:Isothiocyanates; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D013861:Thiocyanates / Q000737:chemistry
D017879:Isothiocyanates; D010455:Peptides; D010761:Phosphorus Radioisotopes; D011506:Proteins; D013861:Thiocyanates; C005441:phenylisothiocyanate