Department of Physical Medicine & Rehabilitation Chang Gung Memorial Hospital, Linkou and Chang Gung University College of Medicine, Tao-Yuan County, Taiwan.
Proteome Sci. 2011 Oct 11;9:65. doi: 10.1186/1477-5956-9-65.
Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly.
The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone.
The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.
蛋白质组学分析常用于鉴定与骨关节炎和其他关节炎疾病相关的膝关节滑液（SF）中的蛋白质或生物标志物。二维凝胶电泳（2-DE）是这些研究中的首选技术。疾病生物标志物通常以低浓度出现，并且可能被高丰度蛋白质掩盖。因此，本研究的主要目的是找到最合适的样品制备方法，以优化可用于开发非骨关节炎膝关节滑液样品参考蛋白质组图谱的 2-DE 凝胶上蛋白质的表达。然后可以将从骨关节炎膝关节滑液中获得的蛋白质组图谱与本研究中获得的参考蛋白质组图谱进行比较，以帮助我们更正确地识别疾病生物标志物。
本研究应用了固定 pH 梯度的 2-DE 蛋白质组学工具。从无骨关节炎的 SF 样本中构建了总共 12 个 2-DE 凝胶图像。在这些样本中，有 3 个未经过任何样品制备方法处理，3 个用丙酮处理，3 个用 2-DE 清洁试剂盒处理，3 个用丙酮和 2-D 清洁试剂盒组合处理，然后再进行 2-DE 分析。使用 PDQuest Basic 8.0.1 分析软件分析凝胶图像。将感兴趣的蛋白斑点从凝胶中取出，通过质谱进行鉴定。计算得到 SF 总蛋白浓度为 21.98±0.86mg/mL。未经处理的 SF 样本在 2-DE 凝胶图像上检测到 456±33 个蛋白斑点。经丙酮处理的 SF 样本检测到 320±28 个蛋白斑点，2-D 清洁试剂盒处理的 SF 样本检测到 413±31 个蛋白斑点，丙酮和 2-D 清洁试剂盒联合处理的样本检测到 278±26 个蛋白斑点 2-DE 凝胶图像。与未经处理的 SF 样本和用丙酮处理的 SF 样本相比，用 2-D 清洁试剂盒处理的 SF 样本在 2-DE 凝胶图像上显示出更清晰的同工型表现，并且 haptoglobin、载脂蛋白 A-IV、前列腺素 D 合酶、alpha-1B-糖蛋白和 alpha-2-HS-糖蛋白等低丰度蛋白质的强度也有所增加。
在进行 2-DE 分析时，丙酮沉淀法和丙酮与 2-DE 清洁试剂盒的联合处理效果都不适合作为 SF 样品的制备方法，因为这两种方法都会导致蛋白质的强度和数量显著减少。另一方面，2-D 清洁试剂盒处理的 SF 样本在 2-DE 凝胶上显示出更清晰的同工型，并且 haptoglobin、载脂蛋白 A-IV、前列腺素 D 合酶、alpha-1B-糖蛋白和 alpha-2-HS-糖蛋白等低丰度蛋白质的强度更高。因此，建议在进行与膝关节骨关节炎相关的相关生物标志物的蛋白质组学研究之前，先用 2-D 清洁试剂盒等蛋白质清洁产品处理 SF 样本。
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