RESUMO
We separated proteins of the middle silk gland through high resolution two-dimensional polyacrylamide gel electrophesis, and identified the high expressional proteins using MALDI-TOF-MS and analyzed the PMF in protein database by GPMAW( General Protein/Mass Analysis for Windows) . The protein database was forecasted through silkworm genome database. More than 500 spots were obtained from each gel by silver stain and more than 100 protein spots were obtained from gel by Coomassie brilliant blue stain. Most of them were distributed in the area from 15kD to 90kD with pH 3.5-7. Among the 25 Coomassie brilliant blue stained spots identified by MALDI-TOF-MS, more than 60% have relatively strong signal. Comparing with the result of using Mascot, the method using PMF database of silkworm not only can identify some known proteins, but also can identify some unknown proteins that have been forecasted in silkworm genome database. Accordingly, we founded a complete set of method that fit for researching proteome of silkworm.
Assuntos
Animais , Sequência de Aminoácidos , Bombyx , Metabolismo , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Concentração de Íons de Hidrogênio , Proteínas de Insetos , Química , Dados de Sequência Molecular , Peso Molecular , Proteoma , Química , Proteômica , Métodos , Seda , Metabolismo , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
High resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was applied to investigate the fat body of silkworm, Bombyx mori. 722 spots were obtained by silver stain from 18 cm, pH 3-10 gel. Most of them were distributed in the area from 15 kD to 90 kD with pH 4 - 8. The matrix-assisted laser desorption ionization time of flight mass spectrometry were applied for identifying the major spots of the 2D map. A total of 41 spots were excised to identify by a combination of MALDI-TOF MS after digested with trypsin. The result showed among the 34 proteins identified, plenty of them were involved in metabolism and immunity. Additionally, Heat shock proteins, 30K proteins and Actin were also detected in the fat body of silkworm. The result will provide a useful tool for understanding the role of fat body in silkworm.