Berger K, Wissmann D, Ihling C, Kalkhof S, Beck-Sickinger A, Sinz A, Paschke R, Führer D
III Medical Department, University of Leipzig, Ph.-Rosenthal-Str. 27, D-04103 Leipzig, Germany.
Mol Cell Endocrinol. 2004 Nov 30;227(1-2):21-30. doi: 10.1016/j.mce.2004.08.001.
Thyroid tumorigenesis involves qualitative and quantitative changes in protein expression, which can be comprehensively studied by proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed proteins. This is due to the limited linear range of most available protein stains, i.e. silver and Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made ruthenium based fluorescent stain (ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform proteome analysis of nodular thyroid disease. We first compared the properties of RuBPs with two highly sensitive protein stains: (1) silver staining and (2) the commercially available fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to Sypro Ruby and silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to Sypro Ruby and 500 times broader than the dynamic range of silver stain), low costs ( 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs stain to 2D gels (pH 4-7) of four benign thyroid nodules and normal thyroid tissue. We were able to detect approximately 1800 protein spots/gel in our thyroid samples. Quantitative changes in protein expression levels of at least 20-42 proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain is a highly sensitive, reliable and inexpensive tool for quantitative proteome analysis in thyroid nodular disease.
甲状腺肿瘤发生涉及蛋白质表达的定性和定量变化，可通过蛋白质组分析进行全面研究。然而，蛋白质组学的技术瓶颈之一仍然是一种可靠、灵敏且廉价的差异表达蛋白质定量方法。这是由于大多数现有的蛋白质染色剂（即银染和考马斯亮蓝）线性范围有限，以及市售荧光染色剂成本高昂。在本文中，我们描述了使用实验室自制的基于钌的荧光染色剂（钌（II）三（二磺酸邻菲罗啉）（RuBPs））进行甲状腺结节疾病蛋白质组分析的经验。我们首先将RuBPs的特性与两种高灵敏度蛋白质染色剂进行了比较：（1）银染和（2）市售荧光染料Sypro Ruby。我们表明，除了具有与Sypro Ruby和银染相似的高灵敏度染色能力（2 ng）外，RuBPs还具有几个优点，如宽动态范围（与Sypro Ruby相似，比银染的动态范围宽500倍）、低成本（每块凝胶0.03美元）以及与质谱的出色兼容性。然后，我们将这种廉价的RuBPs染色剂应用于四个良性甲状腺结节和正常甲状腺组织的二维凝胶（pH 4 - 7）。我们在甲状腺样本中能够检测到每块凝胶约1800个蛋白质点。与同一患者的正常甲状腺组织相比，在良性结节中发现至少20 - 42种蛋白质的表达水平有定量变化。差异表达的斑点通过纳升液相色谱 - 傅里叶变换离子回旋共振质谱和基质辅助激光解吸电离飞行时间质谱进一步表征。总之，我们证明新型荧光钌（II）三（二磺酸邻菲罗啉）染色剂是用于甲状腺结节疾病定量蛋白质组分析的高灵敏度、可靠且廉价的工具。
