This chapter discusses the identification of phosphorylation sites in peptides using a microsequencer. As examples of protein phosphorylation proliferate, identifying the specific phosphorylated residues in peptides becomes a more frequent task. Analysis of phosphorylation sites has not kept pace with advances in amino acid sequencing in large part because the standard Edman degradation procedures do not yield stable phenylthiohydantoin (PTH) derivatives of phosphoserine or phosphothreonine residues; the derivatives of Edman degradation breaking down during the acid cleavage step to release inorganic phosphate. Quantitation of the amount of free inorganic phosphate measures the cumulative release of phosphate from phosphoserine or phosphothreonine residues up to the interrupt cycle and can be used to judge which residues were phosphorylated. The equipment required includes a pair of scissors, gas-phase or pulsed liquid-phase microsequencer, and a means for separating and quantitating phosphate and phosphopeptide.
ASJC Scopus subject areas
- Molecular Biology