Identification of Phosphorylated Amino Acid Residues During Gas Phase Sequencing

Abstract

Covalent phosphory lation is one of the most cannon post-translational modifications of proteins. Identification of the locations of the phosphorylation sites is essential to explore protein kinase specificity and to understand the effects of phosphorylation on biological function. A difficulty encountered in determining phosphorylation sites is that the phosphate covalently associated with serine or threonine residues is released as inorganic phosphate during Edman degradation of these phosphorylated residues. Several attempts have been made to overcome this difficulty. They include high voltage electrophoretic analysis of peptides after manual Edman degradation (1,2), coversion of the phosphorylated residue into other stable derivatives (3,4), and fast atom bombardment mass spectrometry (5). However, all these methods have important limitations. The most cannon method actually used forlocating phosphoryla-tion sites is to measure the P-phosphate in the eluate of each Edman cycle in the spinning-cup or solid-phase sequencer using radioactive peptide (6,7). The problem with this method is that the phosphate released from the phosphorylated residues is difqqult to extract from the sequencer, resulting in low 32P recovery and tailing into the following cycles. Assignment of phosphorylation sites is difficult due to the low 32P recovery, especially for peptides containing multiple potential phosphorylation sites. Furthermore, in the gas phase sequencer, almost no 32P can be detected in the eluate and virtually all of the phosphate remains in the sample filter. In this study, we have developed a simple procedure to identify phosphorylation sites using a gas phase sequencer.KeywordsPhosphorylation SiteEdman DegradationPhosphate ReleaseFast Atom Bombardment Mass SpectrometryStable DerivativeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.