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Identification of phosphotyrosine residues in peptides by high performance liquid chromatography on-line derivative
1Howard Hughes Medical Institute, University of California, San Francisco.
Abstract:
A method has been developed for the detection of phosphotyrosine residues in peptides based on reversed phase-high performance liquid chromatography (RP-HPLC) on-line spectral analysis. It has been found that tyrosine-containing peptides show a hypsochromic shift of the aromatic absorbance maximum when the tyrosine is phosphorylated. Subsequent second-order derivative spectra likewise reveal a hypsochromic shift of the corresponding minima of the phosphotyrosine residues compared to the unmodified tyrosine. This method allows mapping of tyrosine phosphorylation sites in proteins after cleavage into smaller peptides and separation and on-line spectral analysis of the latter by RP-HPLC. It furthermore provides a useful way for the characterization of synthetic phosphotyrosine-containing peptides.