Protein-DNA arrays as tools for detection of protein-protein interactions by mass spectrometry.
Chembiochem
; 14(1): 92-9, 2013 Jan 02.
Article
en En
| MEDLINE
| ID: mdl-23208955
Analysis of multiple protein-protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein-DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt-DNA hybrid molecules described here are used for DNA-directed immobilization on glass- and silica-based microarrays. Methods for the detection of protein-DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Espectrometría de Masas
/
ADN
/
Análisis de Secuencia por Matrices de Oligonucleótidos
/
Mapeo de Interacción de Proteínas
/
Análisis por Matrices de Proteínas
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2013
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Alemania