RESUMEN
Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Membrana Celular/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Octoxinol/química , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Ratas , Reproducibilidad de los Resultados , Solubilidad , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.