Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates.
Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.
van der Geer
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P
Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.
Hunter
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T
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification*; D013379:Substrate Specificity
D007202:Indicators and Reagents; D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D011506:Proteins; D010450:Endopeptidases