Landmark mapping: a general method for localizing cysteine residues within a protein.
We describe a general method to locate the positions of cysteine residues relative to the amino terminus of a protein, using a modified chemical cleavage of the polypeptide backbone at cysteine. The cleavage reaction introduces the carbon atom of 14CN into the carboxyl-terminal fragment produced at each cleavage of the polypeptide chain. Peptides containing the amino terminus of the intact protein are not labeled; all other peptides are labeled at their amino termini. Partial cleavage of a protein followed by gel electrophoresis and autoradiography identifies a ladder of unlabeled peptides that maps positions of the cysteine residues relative to the protein amino terminus. To map individual proteins present in a complex mixture, the polypeptides are cyanolated in solution with 14CN, and the modified proteins are separated by discontinuous SDS/PAGE. The gel is stained, and the desired protein is excised, cleaved at cysteine within the gel slice, and mapped in the second dimension by gel electrophoresis. These techniques are demonstrated with proteins of known sequence containing from zero to five cysteine residues. The cysteine "landmark map" should be particularly useful in locating protein modifications, in questions of protein similarity, and in mapping functional domains. A strategy is also presented for locating other residues in the polypeptide, for which specific cleavage methods exist.
Nefsky
B
B
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
Bretscher
A
A
D000199:Actins / Q000032:analysis; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D003402:Creatine Kinase / Q000032:analysis; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification / Q000648:ultrastructure*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D011506:Proteins; D003402:Creatine Kinase; D003545:Cysteine