Strategies for nonradioactive methods in the localization of phosphorylated amino acids in proteins.
Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.
Meyer
H E
HE
Institut für Physiologische Chemie I, Abteilung Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, Germany.
Eisermann
B
B
Heber
M
M
Hoffmann-Posorske
E
E
Korte
H
H
Weigt
C
C
Wegner
A
A
Hutton
T
T
Donella-Deana
A
A
Perich
J W
JW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000596:Amino Acids; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine