Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes
Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes
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DOI：
10.1002/pmic.200300439
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发表时间：
2003-07-01
期刊：
影响因子：
3.4
通讯作者：
Tonge, R
中科院分区：
文献类型：
--
作者：
Shaw, J;Rowlinson, R;Tonge, R
二维差示凝胶电泳法(2-D DGE)能够提高检测蛋白质差异的置信度。然而，由于最小标记的性质，其中只标记了给定蛋白质的大约5%，不能直接切除斑点用于质谱仪(MS)分析，并且可以进一步提高检测灵敏度。Amersham生物科学公司开发了第二套CyDye(TM)dige Cy(TM)3和Cy5染料，旨在通过饱和标记半胱氨酸残基来克服这些限制。评估了这些染料的灵敏度和动态范围，它们作为多重试剂的可用性，以及从饱和标记凝胶中直接挑选用于MS分析的可能性。饱和标记染料的灵敏度高于其最小标记染料、银染剂和SyproRuby，然而，所产生的二维斑点图案与未标记或最小标记蛋白质的斑点图案显著不同。这些染料被发现是有用的多重试剂，尽管观察到一种染料比另一种染料优先标记蛋白质，但通过实验设计进行了控制。从直接从饱和标记凝胶中切下的材料中成功地获得了蛋白质身份，消除了对染色后制备凝胶的需要。
Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye(TM) DIGE Cy(TM)3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.