RESUMEN
Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.
Asunto(s)
Cromatografía de Afinidad/métodos , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/análisis , Proteómica/métodos , Bases de Datos Factuales , HumanosRESUMEN
The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.
Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meduloblastoma/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor trkA/metabolismo , Transducción de Señal , Adolescente , Animales , Proteínas Portadoras/genética , Muerte Celular , Línea Celular Tumoral , Niño , Preescolar , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Neoplasias/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteómica , Receptor trkA/genéticaRESUMEN
Affinity purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear, however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is useful for reducing sample complexity prior to MS analysis and increases peptide and protein coverage of complex samples. Here, we monitored the effects of upstream protein or peptide separation techniques on typical mammalian AP-MS samples, generated by FLAG affinity purification of four baits with different biological functions and/or subcellular distribution. As a first separation step, we employed SDS-PAGE, strong cation exchange LC, or reversed-phase LC at basic pH. We also analyzed the benefits of using an instrument with a faster scan rate, the new TripleTOF 5600 mass spectrometer. While all multidimensional approaches yielded a clear increase in spectral counts, the increase in unique peptides and additional protein identification was modest and came at the cost of increased instrument and handling time. The use of a high duty-cycle instrument achieved similar benefits without these drawbacks. An increase in spectral counts is beneficial when data analysis methods relying on spectral counts, including Significance Analysis of INTeractome (SAINT), are used.