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Identification of phosphorylation sites by Edman sequencing |
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A number of phosphorylated proteins are available in quantities too small for conventional protein chemistry. However the position of phosphorylation sites in such proteins can be determined. The protein is digested with a proteinase of known specificity, most commonly trypsin, and the peptides are isolated. Two dimensional thin layer chromatography and reverse phase chromatgraphy are the normal methods for peptide purification. The peptide should be supplied in less than 100 microliters of volatile solvent; dilute formic/acetic acid used for TLC has been satisfactory. It is preferable to design sample preparation to avoid drying samples because drying may cause loss of sample. The peptide is covalently coupled to a modified PVDF membrane and washed to remove unbound peptide. Coupling efficiencies range from 30% to 95%. The sample is sequenced with a cycle designed for phosphopeptides. The cycle uses trifluoroacetic acid to dissolve phosphoamino acids and inorganic phosphate which are delivered to a fraction collector and counted. There is no identification of amino acids with this technique. The number of counts needed depends on how well the peptide can be removed from the tube in which it was supplied, how well it couples, how well it sequences and the position of the phosphorylation site. We suggest supplying at least 1,000 c.p.m., although a few samples gave data with less counts. The most distant phosphorylation site for which we have obtained good data is position 16. References
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