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Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions
Published on: April 21, 2016
Eludir los problemas causados por la diversidad de proteínas en microarrays: implicaciones para las redes de
Andrew Gordus1, Gavin MacBeath
1Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Journal of the American Chemical Society
|October 19, 2006
Resumen
Los microarrays de proteínas pueden representar de manera inexacta las interacciones de las proteínas debido a variaciones en las propiedades físicas. La obtención de curvas de unión de saturación proporciona datos precisos y cuantitativos para un análisis fiable de la red de interacción de proteínas.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Molecular Biología Molecular
- Biología de Sistemas Biología de Sistemas.
Sus antecedentes:
- Los microarrays de proteínas son herramientas de alto rendimiento para estudiar las interacciones proteína-proteína.
- Las variaciones en las propiedades físicas de las proteínas pueden sesgar los resultados de ensayos estandarizados como los microarrays.
- Los análisis simples basados en umbrales pueden conducir a redes de interacción binaria engañosas.
Objetivo del estudio:
- Para investigar la variabilidad de los dominios de interacción de proteínas en microarrays.
- Para evaluar el impacto de las variaciones de la densidad de la superficie en los datos de interacción.
- Identificar métodos para obtener datos cuantitativos precisos de las micro matrices de proteínas.
Principales métodos:
- Impresión de dominios monoméricos Src homología 2 (SH2) en concentraciones uniformes.
- Realización de ensayos de unión basados en umbrales con fosfopéptidos etiquetados con fluorescencia.
- Obtención de curvas de enlace de saturación para las interacciones proteína-péptido.
Principales resultados:
- Los dominios monoméricos SH2 mostraron hasta 50 veces la variación en la densidad de la superficie activa cuando se imprimió a la misma concentración.
- Los ensayos basados en umbrales produjeron datos engañosos de interacción biofísica.
- Las constantes de disociación de equilibrio derivadas de las curvas de saturación eran independientes de la densidad de la superficie.
Conclusiones:
- Las variaciones de las propiedades físicas de las proteínas tienen un impacto significativo en los estudios de interacción basados en microarrays.
- El análisis cuantitativo utilizando curvas de unión de saturación supera los sesgos de densidad de superficie.
- Los futuros estudios de redes de interacción de proteínas a gran escala deberían priorizar la adquisición de datos cuantitativos.
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