Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis.
Proteomics
; 17(9)2017 May.
Article
en En
| MEDLINE
| ID: mdl-28252257
ABSTRACT
Large clostridial toxins mono-O-glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [14 C]glucosylation, sequential [32 P]ADP-ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS-based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo-2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R-Ras2.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
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Toxinas Bacterianas
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Proteínas de Unión al GTP Monoméricas
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Proteómica
Límite:
Humans
Idioma:
En
Año:
2017
Tipo del documento:
Article