Determination of the primary structure of homologous proteins by sequence analysis of peptide mixtures

We propose a rapid method to determine the primary structure of a protein knowing the sequence of a homologous protein. The method consists in submitting both the reduced and alkylated proteins to an enzymatic or chemical hydrolysis and performing the sequence analysis of the peptide mixtures. The assessment of the unknown sequence and the degree of identity of the two proteins are reached by comparing the two sequence analyses. The sequences of all the possible peptides present in the two mixtures are reconstructed and the differences in the two sequences are determined. If necessary, the differences can be confirmed by performing a mass spectrometric analysis of the two mixtures. We used this procedure on two homologous proteins of known sequence to furnish an application example of the method.