Exploring the nature of inclusion bodies by MALDI mass spectrometry using recombinant proinsulin as a model protein.
Int J Biol Macromol
; 139: 647-653, 2019 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-31356950
ABSTRACT
The present study deals with mass spectrometric investigation to characterize the nature of proinsulin in inclusion bodies. Various derivatives of human proinsulin were cloned, expressed in E. coli and inclusion bodies prepared under weak acidic conditions (pHâ¯6.5), which protected the native thiols. Non-reductive PAGE showed that proinsulin migrated as monomer (approximately 10â¯kDa). MALDI-MS protocol was developed for the direct analysis of proinsulin derivatives in inclusion bodies. It was found that the masses of the derivatives corresponded to polypeptides containing six cysteines in reduced form. Iodoacetamide or iodoacetic acid treatment of proinsulin inclusion bodies, in suspension under non-reducing conditions and without any chaotropic agents, showed six alkylations, suggesting that these cytoplasmic aggregates were assembled from reduced monomers, with their -SH groups pointing towards hydrophilic surface. The MALDI analysis of inclusion bodies was extended to a proinsulin derivatives labelled with 13C and 15N giving an excellent agreement between experimental and theoretical masses. These mass spectrometric studies also provide early information about post-translational modification as evident in one of the derivatives MTRR-pi showing N-terminal cleavage of methionine. This shows the potential value of the protocol for the accurate analysis of polypeptides, expressed as inclusion bodies, prior to undertaking further purification.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Proinsulina
/
Proteínas Recombinantes
/
Corpos de Inclusão
/
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
Int J Biol Macromol
Ano de publicação:
2019
Tipo de documento:
Article