Comparison of urinary exosomal protein solubilization methods for two-dimensional gel electrophoresis

Asami Kosaka, Akinobu Nakayama, Iku Yamaguchi, T. Kasama, M. Totsuka, K. Shiba
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Abstract

Background: Urinary exosomal proteins have recently emerged as important candidates for elucidating the mechanisms underlying physiological events and disease-related metabolism in the kidney. Here, we evaluated standard sample preparation methods for two-dimensional gel electrophoresis (2DE) to determine which one yielded the maximum protein recovery from urinary exosomes for protein identification.Materials and Methods: Urinary exosomes were purified from a healthy subject by using ultracentrifugation. The final pellets were dissolved with PBS or RIPA buffer. After being desalted, these exosomal protein solutions were each treated with 1 of 4 rehydration buffers (Rbs) containing detergents in the following formulations: CHAPS (Rb1), CHAPS and Triton X-100 (Rb2), dodecyl maltoside (Rb3), and ASB-14 (Rb4).Results: For all Rbs, a much greater number of protein spots was detected in the samples isolated with RIPA than with PBS. Only minor differences were observed in the number of protein spots for Rb1-3. The largest protein spots were detected using the combination of RIPA buffer and Rb4; however, the background on the 2DE gel was high in the region of >66 kD and at the lower pH values. For all combinations, the co-precipitation of the urinary Tamm-Horsfall protein masked the protein spots in the 66-100 kD region.Conclusion: For extracting a large number of proteins with a relatively clear background on silver-stained 2DE gels, the optimal exosomal protein-dissolving buffer is RIPA buffer. All of the evaluated Rbs, except for the one containing ASB-14 as a detergent, is suitable for solubilizing exosomal proteins on 2DE.
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尿外泌体蛋白二维凝胶电泳增溶方法的比较
背景:尿外泌体蛋白最近成为阐明肾脏生理事件和疾病相关代谢机制的重要候选者。在这里,我们评估了二维凝胶电泳(2DE)的标准样品制备方法,以确定哪一种方法可以从尿外泌体中获得最大的蛋白质回收率,用于蛋白质鉴定。材料和方法:采用超离心法从健康受试者尿液中纯化尿外泌体。最后的微球用PBS或RIPA缓冲液溶解。脱盐后,这些外泌体蛋白溶液分别用4种复水缓冲液(Rbs)中的1种进行处理,其中含有以下配方的洗涤剂:CHAPS (Rb1), CHAPS和Triton X-100 (Rb2),十二烷基麦芽糖苷(Rb3)和ASB-14 (Rb4)。结果:对于所有的Rbs,在用RIPA分离的样品中检测到的蛋白点数量比用PBS分离的要多得多。Rb1-3的蛋白斑点数量差异不大。用RIPA缓冲液和Rb4联合检测最大的蛋白斑点;然而,在>66 kD区域和较低的pH值下,2DE凝胶的本底值较高。对于所有组合,尿Tamm-Horsfall蛋白的共沉淀掩盖了66-100 kD区域的蛋白斑点。结论:对于在银染2DE凝胶上提取大量背景相对清晰的蛋白质,最佳的外泌体蛋白溶出缓冲液为RIPA缓冲液。除了含有ASB-14作为去污剂的Rbs外,所有评价的Rbs都适合溶解2DE上的外泌体蛋白。
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