Abstract
Characterization of 5 to 25 pmol of purified proteins by tryptic peptide mapping has been accomplished using the Bolton-Hunter reagent ( 125I-3-[4-hydroxyphenyl]propionic acid N-hydroxysuccinimide ester). Radioacylation is followed by reaction with unlabeled ester and reductive methylation to ensure resistance of lysyl residues to trypsinization. Reduced and alkylated proteins are analzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsinized from individual gel slices, and mapped two-dimensionally on thin layers. The method permits peptide mapping of proteins with specific activities of 1 to 2 × 10 4 cpm/ng, results in more spots (and often more structural information) than direct iodination procedures, and can be used for characterization of proteins that could not be biosynthetically labeled.
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