Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine.

Abstract

Many proteins cannot be directly sequenced by Edman degradation because they have a blocked N-terminal residue. A method is presented for deblocking such proteins when the N-terminal residue is N-acetylserine (which occurs frequently in eukaryotic proteins) or N-acetylthreonine. The method has been applied successfully to the determination of the N-terminal amino acid sequence of human, bovine, and rat parathymosins. Prothymosin a and other blocked proteins and peptides were also readily deblocked and sequenced by this procedure. It is proposed that the mechanism ofthe deblocking reaction involves an acid-catalyzed N -0 shift of the acetyl group followed by a fl-elimination.Although notable improvements have been made recently in the instrumentation' available for automated sequencing of proteins and peptides, many proteins still present a challenging problem to investigators who attempt to determine their sequence. One problem frequently encountered is that the N-terminal residue is modified in such a way that it does not react with the Edman reagent phenyl isothiocyanate. For

Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis

“…After treatment of the e lzyme with TFA/methanol, Edman degradation revealed the s~quence Ala-Asp-Lys-which shows that the method also orks for acetylated N-terminal residues other than Ser and q hr. This finding implies that the mechanism involved is not a smple acid-catalyzed N ~ O shift of the acetyl group followed b'¢ 13-elimination which has been suggested for deacetylation c" N-terminal Ser and Thr employing neat TFA [5], but rather a more specific reaction involving the methanol.…”

“…It involves relatively little handling, and no thermostated incubator is necessary. The significant advantage is the low yield of internal peptide bond cleavage, also for large polypeptides and proteins, a major ditticulty encountered with other pro- tc,cols [5], which is far more important than the somewhat low ivitial yield, in allowing interpretation of the amino acid seq tences for quite long segments after deblocking.…”

“…Drawbacks with that technique are high protein consumption, leag handling times, and maintained inaccessibility of the N .terminal peptide to Edman degradation. To deblock the N .terminal fragment, both enzymatic and chemical protocols hz,ve been reported [2,4,5]. Among chemical cleavages an inte'esting approach employs neat trifluoroacetic acid at elew, ted temperature (45-65°C) with incubation overnight or fc~ 3 days [5].…”

“…To deblock the N .terminal fragment, both enzymatic and chemical protocols hz,ve been reported [2,4,5]. Among chemical cleavages an inte'esting approach employs neat trifluoroacetic acid at elew, ted temperature (45-65°C) with incubation overnight or fc~ 3 days [5]. However, for large polypeptides and proteins tin s gives too high a background for clear sequence interpreta :ion [5] because of internal peptide bond cleavage due to low s[ecificity in the deacetylation chemistry employed.…”

Isolation and Amino Acid Sequences of Two Trypsin Inhibitors from the Seeds of Bitter Gourd (Momordica charantia)

“…Deacetylation of the acetyl group at N-terminal serine of peptide was done by treatment of the peptide (10 nmol) with TF A vapor in an evacuated sealed tube at 65°C for 16 h by the method of Wellner et al 11) Acetylation of inhibitor was done by the method of Montelaro et al 12) One III of acetic anhydride was added to 100 III of inhibitor (1 mg) solution in 0.3 M phosphate buffer, pH 7.2, and stirred for 30 min. Excess reagents were removed by dialysis against deionized water.…”