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Protocolos de Proteómica de Baja Entrada para Muestras FFPE Capturadas con Láser con Orbitrap Astral DIA-MS
Daniele Musiani1, Alessandro Cuomo2
1European Institute of Oncology (IEO), Mass Spectrometry Unit (MSU), Milan, Italy.
Methods in molecular biology (Clifton, N.J.)
|February 2, 2026
Resumen
Dos nuevos protocolos para microdisección láser (LMD) y espectrometría de masas (MS) permiten el análisis proteómico sensible de diminutas muestras de tejido. Estos métodos minimizan la pérdida de muestras para la investigación detallada de la heterogeneidad tisular en muestras clínicas.
Área de la Ciencia:
- Proteómica
- Biología Molecular
- Bioquímica
Sus antecedentes:
- La microdisección láser (LMD) acoplada a la espectrometría de masas (MS) es vital para analizar la heterogeneidad tisular.
- El análisis de muestras de tejido de baja entrada presenta desafíos significativos en estudios proteómicos.
- Los protocolos existentes pueden no estar optimizados para una pérdida mínima de muestras o para el procesamiento manual.
Objetivo del estudio:
- Presentar dos protocolos optimizados para la preparación de muestras de baja entrada para el análisis LMD-MS.
- Permitir el análisis proteómico sensible y de alta resolución de regiones de tejido microdiseccionadas.
- Proporcionar orientación para el perfilado proteómico reproducible de muestras clínicas.
Principales métodos:
- Desarrollo y optimización de dos flujos de trabajo de preparación de muestras de baja entrada: Preparación Mejorada en Fase Sólida (SP3) y Detergente/Disolvente Orgánico (DDM/ACN).
- Aplicación de protocolos a pequeñas áreas de tejido (hasta 1500 μm² de tejido FFPE de 10 μm de espesor).
- Integración con espectrometría de masas de alta resolución (Orbitrap Astral) utilizando Adquisición Independiente de Datos (DIA).
Principales resultados:
- Los protocolos minimizan la pérdida de muestras, lo que permite el análisis de regiones de tejido muy pequeñas.
- Se logró un perfil proteómico reproducible con una profundidad proteómica sustancial a partir de muestras de baja entrada.
- Se demostró un análisis proteómico sensible y de alta resolución de tejido FFPE microdiseccionado.
Conclusiones:
- Los protocolos SP3 y DDM/ACN desarrollados ofrecen soluciones robustas para LMD-MS de baja entrada.
- Estos métodos facilitan la investigación de la heterogeneidad espacial en muestras clínicas con alta precisión.
- Los protocolos son adecuados para el procesamiento manual, lo que aumenta la accesibilidad para investigadores sin plataformas de automatización.
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