Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format
Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format
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DOI：
10.1002/rcm.1024
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发表时间：
2003-01-01
影响因子：
2
通讯作者：
Nagasu, T
中科院分区：
文献类型：
--
作者：
Katayama, H;Satoh, K;Nagasu, T
凝胶内消化效率的提高对于蛋白质组学中的一维或二维凝胶电泳分离和质谱（MS）分析是非常期望的，因为所得的序列覆盖度和MS信号强度的增加导致蛋白质鉴定的更高置信度。这里描述了一种优化的凝胶内消化系统，结合使用96孔板的高通量格式中的薄凝胶分离和阴性染色。在0.9 mm厚的凝胶上进行负染色和蛋白质分离的组合与更典型的方案（例如银染色和1.0 mm凝胶的组合）相比，显示出凝胶内消化效率的明显改善。此外，当在诸如脱色、洗涤、凝胶收缩和肽提取的过程中通过超声处理而不是摇动板来进行凝胶块的搅拌时，使用96孔板来增加通量不会降低该策略的效率。该程序进行了优化，并应用于识别突触后密度级分的蛋白质; SDS-PAGE分离后，105个蛋白质被确定。版权所有（C）2003约翰威利父子有限公司。
Improvement of in-gel digestion efficiency is highly desirable for one- or two-dimensional gel electrophoretic separation and mass spectrometric (MS) analysis in proteomics, because the resultant increases in sequence coverage and MS signal intensity lead to higher confidence in protein identification. Here an optimized in-gel digestion system, in combination with thin-gel separation and negative staining in a high-throughput format using 96-well plates, is described. The combination of negative staining and protein separation on a 0.9 mm thick gel showed a clear improvement in in-gel digestion efficiency in comparison with the more typical protocols such as the combination of silver staining and a 1.0 mm gel. In addition, the use of 96-well plates to increase throughput did not decrease the efficiency of this strategy when the stirring of the gel pieces in processes such as destaining, washing, gel-shrinking and peptide extraction was performed by sonication instead of shaking the plates. This procedure was optimized and applied to identify proteins of the postsynaptic density fraction; 105 proteins were identified after SDS-PAGE separation. Copyright (C) 2003 John Wiley Sons, Ltd.