Isolation technique and proteomic analysis of the erythrocyte ghosts of red‐eared turtle (Trachemys scripta)
Isolation technique and proteomic analysis of the erythrocyte ghosts of red‐eared turtle (Trachemys scripta)
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DOI：
10.1002/elps.201200243
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发表时间：
2013-01
期刊：
影响因子：
2.9
通讯作者：
Jun Gao;Jianglin Li;C. Feng;Zhaotun Hu;Wenfeng Liu;S. Liang;D. Yin
中科院分区：
文献类型：
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作者：
Jun Gao;Jianglin Li;C. Feng;Zhaotun Hu;Wenfeng Liu;S. Liang;D. Yin
为了对红耳龟Trachemys scripta的红细胞进行蛋白质组学分析，首先开发了一种获得龟红细胞血影（TEG）的方法。使用特殊缓冲液低渗裂解后，用“N”形针迫使红细胞通过注射器，低速匀浆和差速离心，获得高纯度的TEG组分。对分离的 TEG 蛋白进行凝胶内消化处理，通过 SDS-PAGE 或溶液内消化进行分离，然后进行 HPLC 预解离，然后通过 nano-ESI-LC MS/MS 技术进行鉴定。总共鉴定、验证和分类了 169 个 TEG 蛋白。在这些蛋白质中，微管蛋白和位于细胞表面的F型ATP合酶揭示了Trachemys scripta在缺氧和低温暴露下的超耐受性的重要信息。总之，本研究不仅提供了分离高质量TEG的方法和全面表征TEG蛋白的数据集，而且为全有核红细胞的蛋白质组学研究提供了工具，从而为探索海龟在恶劣环境中的超耐受机制开辟了新的研究领域。
To proceed proteomic analysis of erythrocyte of the red‐eared turtle Trachemys scripta, a method for obtaining turtle erythrocyte ghosts (TEG) was first developed. After hypotonic lysis using a special buffer, forcing the erythrocyte through the syringe with an “N”‐shaped needle, applying low speed homogenizing and differential centrifugation, highly purified TEG fractions were obtained. The isolated TEG proteins were treated with in‐gel digestion separated by SDS‐PAGE or in‐solution digestion followed by HPLC predissociation, and then identified by nano‐ESI‐LC MS/MS techniques. A total of 169 TEG proteins was identified, validated, and categorized. Among these proteins, tubulins, and cell‐surface‐located F‐type ATP synthase revealed important information into the super tolerance of Trachemys scripta in anoxia and low temperature exposure. Altogether, this study not only provided a method to isolate high quality TEG and a dataset of comprehensive characterization of TEG proteins, but also provides a tool for proteomic research of all nucleated red blood cells, and thus opened a new research field for exploring the mechanisms of super tolerance of turtles in harsh environment.