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

Abstract

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 identify 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.

Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

“…These difficulties have been greatly reduced using modern pulsed-liquid phase sequencers, since the carryover is a well quantifiable phenomenon and, despite the yield problems, the identification of several amino acid residues at each step requires just a little care and experience. [43][44][45][46][47][48][49][50][51][52][53][54] Thus, digesting a protein of known sequence by any agent, it is possible to assess the sequence of each fragment present in the mixture and evaluate its amount. Although the absolute quantification is underestimated due to the initial coupling yield of Edman degradation (60-65%), the data can be compared and supply quantitative information since they suffer for a similar error.…”

“…In the last years, the automatic sequence analysis of peptide mixtures by modern sequencers equipped on-line with phenylthiohydantoin (PTH) amino acid analyzers has been usefully used to characterize proteins. Different strategies in assessing protein sequences have been reported [43][44][45][46][47][48][49] together with methods for determining the hydrolysis pathway of polypeptides by unknown digesting agents. [50][51][52][53] These approaches, which have been recently reviewed, 54 do not require any purification step and are based upon the sequence determination of the fragments in mixture; moreover, sequence data being quantitative, the amount of each fragment can be evaluated.…”

“…[50][51][52][53] These approaches, which have been recently reviewed, 54 do not require any purification step and are based upon the sequence determination of the fragments in mixture; moreover, sequence data being quantitative, the amount of each fragment can be evaluated. [43][44][45][46][47][48][49][50][51][52][53][54] The method can also be used to get rapid information on the tertiary structure of proteins in solution. In fact, if a native protein of known sequence is digested by a specific protease, it is possible to follow the kinetics of the hydrolysis at the recognized sites by quantitative sequence analysis of the mixture products at different digestion times.…”

Analytical techniques and computer algorithms combined for the rapid characterization of structural peptide and protein features

“…With this approach, the authors determined the structural differences between two a-amylase isoinhibitors from wheat. Two residues were deleted in one sequence, and just one amino acid of 123 was substituted (26,27).…”

Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures

“…In the last years, the automatic sequence analysis of peptide mixtures by modern sequencers equipped on‐line with phenylthiohydantoin (PTH) amino acid analyzers has been usefully used to characterize proteins. Different strategies in assessing protein sequences have been reported43–49 together with methods for determining the hydrolysis pathway of polypeptides by unknown digesting agents 50–53. These approaches, which have been recently reviewed,54 do not require any purification step and are based upon the sequence determination of the fragments in mixture; moreover, sequence data being quantitative, the amount of each fragment can be evaluated 43–54.…”