Peptide mapping of S-carboxymethylated hair and feather proteins using two-dimensional electrophoresis

Abstract

The aim of this work was to develop a two-dimensional electrophoretic method for the multiple simultaneous peptide mapping of a broad spectrum of human and animal hair and avian feather proteins. The 14C labelled S-carboxymethylated proteins of hair and feather were separated in one dimension by sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis and stained with Coomassie Blue. Each of the gel lanes containing the separated hair or feather proteins from one individual was cut and transferred at right angles on to a second slab gel. A solution of trypsin (5 μg/ml) in stacking gel buffer was poured on to the gel lane. Partial proteolysis of the hair or feather proteins proceeded in situ while the stacking gel buffer set. The second dimension of electrophoresis followed by staining and/or fluorography showed a characteristic pattern of peptides of proteins in the form of spots derived from each individual protein, leaving undigested proteins well separated on the diagonal. The protease digestion pattern was reproducible and characteristic of each sample. We were able to establish a finer peptide signature, for individual samples containing a mixture of many proteins, than was hitherto possible with previously published two-dimensional electrophoretic techniques. It was concluded that this technique may be of use in future evolutionary, ontogenetic and forensic studies using hair and feathers as a biological source material.