Partial proteolysis improves the identification of the extracellular segments of transmembrane proteins by surface biotinylation.
Sci Rep
; 10(1): 8880, 2020 06 01.
Article
em En
| MEDLINE
| ID: mdl-32483232
ABSTRACT
Transmembrane proteins (TMP) play a crucial role in several physiological processes. Despite their importance and diversity, only a few TMP structures have been determined by high-resolution protein structure characterization methods so far. Due to the low number of determined TMP structures, the parallel development of various bioinformatics and experimental methods was necessary for their topological characterization. The combination of these methods is a powerful approach in the determination of TMP topology as in the Constrained Consensus TOPology prediction. To support the prediction, we previously developed a high-throughput topology characterization method based on primary amino group-labelling that is still limited in identifying all TMPs and their extracellular segments on the surface of a particular cell type. In order to generate more topology information, a new step, a partial proteolysis of the cell surface has been introduced to our method. This step results in new primary amino groups in the proteins that can be biotinylated with a membrane-impermeable agent while the cells still remain intact. Pre-digestion also promotes the emergence of modified peptides that are more suitable for MS/MS analysis. The modified sites can be utilized as extracellular constraints in topology predictions and may contribute to the refined topology of these proteins.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Proteínas de Membrana
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Hungria