Quantitative solid-phase Edman degradation for evaluation of extended solid-phase peptide synthesis.
Quantitative solid-phase Edman degradation was used for the amino acid sequence analysis of synthetic peptidyl-resins prepared by the Merrifield solid-phase procedure. A model peptide, Ala-[3H]Pro-Ala-Gly-Phe-Ala-Gly-, was synthesized on a solid support and was sequenced to measure the efficiency of the solid-phase sequencing protocol used. An average of 92% of the first four residues was removed from the peptidyl-resin as indicated by subtractive amino acid analysis. Quantitation of the radioactive proline residue at cycle 2 revealed that it was efficiently recovered both from the acid conversion procedure (99%) and also following high-pressure liquid chromatography of the phenylthiohydantoin (Pth) amino acid (88%). In order to facilitate identification and quantification of the side chain protected Pth amino acids, we prepared these derivatives and characterized them by high-pressure liquid chromatography. Thereafter, by the use of solid-phase Edman degradation as an analytical procedure, the synthesis of residues 2-118 of the heavy-chain variable region (VH) of a homogeneous rabbit antibody was undertaken. At 10-15-residue intervals during the solid-phase synthesis, samples of peptidyl-resin were removed from the synthesis vessel and sequenced. When gross synthetic errors caused by deletion of amino acids residues were detected, the solid-phase synthesis was terminated and restarted by using modified protocols. A 117-residue peptidyl-resin was prepared finally which possessed the desired amino acid sequence as indicated by a series of solid-phase Edman degradation experiments. In the final degradation experiment on the 117-residue peptidyl-resin, a 92% efficiency for the automatic Edman reaction was measured ([3H]Leu, penultimate amino-terminal residue). We have found two advantages for the concurrent use of solid-phase Edman degradation during an extended solid-phase synthesis: (1) on the basis of the level of error due to incomplete incorporation of amino acids, the solid-phase assembly could be terminated in favor of restarting the synthesis, hence avoiding further work on a defective product and (2) direct verification of incorporation of amino acids, which during acid hydrolysis are destroyed (Cys, Trp) or are deamidated (Asn, Gln), is possible by high-pressure liquid chromatography of the corresponding Pth derivatives.