Cleavage and Identification of Proteins:A Modified Aspartyl-Prolyl Cleavage

摘要

We have developed a method for rapidly cleaving and identifying proteins electroblotted onto poly(vinylidene difluoride)membranes. Cleavage is performed with 10% acetic acid in 7 M guanidine chloride at pH 2.5 for 1 h at 90 deg C, resulting in fragmentation primarily at aspartylprolyl onds. Peptides resulting from non-Asp-Pro cleavage are N-terminally blocked by reaction with orthophthalaldehyde(OPA)prior to automated Edman degradation. Reaction with OPA afte rcleavage blocks all amino acids containing primary amino groups. Only peptides containing an N-terminal amino acid with a secondary amino group(proline)will be available for reaction with the Edman reagent. The sequences obtained are used for protein database searchin. Using this approach, proteins that are foudn to be N-terminally blocked acan be removed from the sequencer, cleaved with acetic acid, blopcked with OPA, and reapplied to the sequencer. The protein can then be identified from a database search using the sequence mixture obtained.