Towards global analysis of mosquito chorion proteins through sequential extraction, two-dimensional electrophoresis and mass spectrometry.
Towards global analysis of mosquito chorion proteins through sequential extraction, two-dimensional electrophoresis and mass spectrometry.
复制标题
通过顺序提取、二维电泳和质谱对蚊子绒毛膜蛋白进行全局分析。
DOI：
10.1002/pmic.200300536
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发表时间：
2003
期刊：
影响因子：
3.4
通讯作者：
Li,Jianyong
中科院分区：
文献类型：
--
作者：
Yao,Ruiliang;Li,Jianyong
本研究描述了通过二维电泳（2-DE）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）技术分离和鉴定绒毛膜蛋白。由于绒毛膜蛋白的高疏水性，其难以被溶解并吸附到固定化pH梯度条中用于等电聚焦。通过优化绒毛膜蛋白提取和样品应用的应用条件，我们能够溶解大部分绒毛膜蛋白并通过2-DE将其解析。在优化的条件下，通过二维分析可以分辨出700多个蛋白质点。通过胰蛋白酶消化单个蛋白质点，对其消化肽进行MALDI-TOF MS分析，随后对肽质量进行BLAST搜索，初步鉴定出38个蛋白质点。我们的数据表明，分离的绒毛膜的连续提取，溶解的绒毛膜蛋白的2-DE，溶解的蛋白质的凝胶内消化和蛋白酶的MALDI-TOF MS分析是测定蚊子绒毛膜蛋白的有效的总体策略。讨论了该方法用于测定蚊子绒毛膜蛋白的优点及其用于分离和鉴定其他昆虫膜蛋白和绒毛或卵壳蛋白的可行性。
This study describes the separation and identification of chorion proteins through two‐dimensional electrophoresis (2‐DE) and matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) techniques. Due to their high hydrophobicity, chorion proteins are difficult to be solubilized and absorbed into the immobilized pH gradient strip for isoelectric focusing. By optimizing the applied conditions for chorion protein extraction and sample application, we were able to solubilize the majority of the chorion proteins and resolve them by 2‐DE. Under optimized conditions, there are more than 700 protein spots resolved by 2‐D analysis. Trypsin digestions of individual protein spots, MALDI‐TOF MS analysis of their digested peptides, and subsequent BLAST search of peptide masses resulted in the tentative identification of 38 protein spots. Our data show that sequential extraction of the isolated chorion, 2‐DE of the solubilized chorion proteins, in‐gel digestion of the resolved protein and MALDI‐TOF MS analysis of the protein digests is an effective overall strategy towards determination of chorion proteins in mosquitoes. The merits of the method described for the determination of mosquito chorion proteins and its feasibility for the separation and identification of membrane proteins and chorion or eggshell proteins from other insect species are discussed.