A method for the unambiguous identification of tryptophan in automated protein sequence analysis.
Anal Biochem
; 191(1): 169-73, 1990 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-2077939
ABSTRACT
Some widely used standard protocols for the separation of phenylthiohydantoin amino acid derivatives by reverse-phase gradient HPLC do not provide separation of the phenylthiohydantoin derivative of tryptophan (PTH-Trp) from diphenylurea (DPU), a by-product generated during Edman degradation of proteins in variable amounts. Furthermore, PTH-Trp is usually recovered in low yield under typical experimental conditions used with automated sequencing equipment. These factors may compromise the unambiguous assignment of tryptophan residues in automated protein sequence analysis, especially when sequencing is performed at high sensitivity. We devised a reverse-phase HPLC method which allows the separation of DPU and PTH-Trp and therefore the correct assignment of PTH-Trp. The method is based on a modification of the HPLC gradient used to elute and separate all PTH amino acids of interest. With Applied Biosystems Model 477A protein sequencers with on-line PTH amino acid identification, the correct assignment of tryptophan was consistent and reproducible even when sequencing at very high sensitivity (5 pmol).
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Triptofano
/
Proteínas
/
Cromatografia Líquida de Alta Pressão
/
Sequência de Aminoácidos
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
1990
Tipo de documento:
Article