Phosphoamino acid analysis, or PAA, is an experimental technique used in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
to determine which amino acid or acids are
phosphorylated
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, whi ...
in a protein.
Technique
A protein is first phosphorylated using
32P-labeled ATP, usually via an ''in vitro'' kinase assay. Most of the amino acids
in the protein are then hydrolyzed, usually by the use of a strong acid such as
hydrochloric acid
Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid
Acid strength is the tendency of an acid, symbol ...
. These amino acids are then separated using 2-dimensional
thin layer chromatography
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.
Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a t ...
, along with amino acid standards for the three amino acids that are phosphorylated in eukaryotes:
serine
Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
,
threonine
Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated −COOâ ...
, and
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the Gr ...
. These amino acid standards can be visualized on the TLC substrate by exposure to
ninhydrin
Ninhydrin (2,2-dihydroxyindane-1,3-dione) is an organic compound with the formula C6H4(CO)2C(OH)2. It is used to detect ammonia and amines. Upon reaction with these amines, ninhydrin gets converted into deep blue or purple derivatives, which are ...
, which colors the amino acids a visible purple when heated at ~100 °C. The radioactive amino acids can be detected via autoradiography, and an overlay of the two images will show which amino acids are phosphorylated.
References
*Rothberg, P.G., Harris, T.J.R., Nomoto, A., and Wimmer, E. (1978) O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. ''Proc. Natl. Acad. Sci. USA'' 75, 4868-4872.
*Eckhart, W., Hutchinson, M.A., and Hunter, T. (1979) An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. ''Cell'' 18, 925-933.
Protein methods