Source : Proteomics: From Sequence to Function.

Operation method

basic program

Materials and Instruments

Protein samples

Move

Knowing the type of phosphorylated amino acid in a peptide or protein can qualify possible phosphorylation sites and therefore simplify (the identification of phosphorylated residues in a peptide chain. The type of phosphorylated residue is usually determined by phosphoamino acid analysis or phosphoamino acid-specific immunoassays. Phosphoamino acid analysis involves the gas- or liquid-phase hydrolysis of peptide bonds, and this hydrolysis is performed under conditions that leave at least one segment of the phosphate bond intact. Hydrolyzed products of ^32p-labeled phosphoproteins or phosphopeptides are mixed with phosphate amino acid standards and separated by TLC. Unlabeled standards were detected by ninhydrin staining. 32p-labeled amino acids were detected by radioautography. The type of phosphoaminoamine present in the protein is determined by correlation analysis of the stained standard with the radiolabeled species. If a large number of samples are available. Phosphoamino acid types can be determined directly by ninhydrin staining. Phosphoamino acid analysis with phosphopeptides extracted from 2D-PP spectra is particularly useful． These peptides usually contain only one phosphoamino acid type. Knowledge of the nature of the phosphorylated amino acid and other parameters, such as the molecular mass of the peptide and the properties of the enzyme used to cleave the protein, can in some cases identify or at least infer the phosphorylation site in the protein. The type of phosphorylated amino acid can be identified with certainty using the antibody specificity of the particular phosphoamino acid, but this method well not be a substitute for phosphoamino acid analysis. Tyrosine-phosphorylated proteins are easily detected after gel separation by immunoblotting with tyrosine-phosphate antisera, including 4g10 and py20, but also antibodies to bacterial fragments such as RC10.

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