Identification and characterization of cell wall proteins of a toxic dinoflagellate Alexandrium catenella using 2-D DIGE and MALDI TOF-TOF mass spectrometry
首发时间:2011-04-06
Abstract:Cell wall is an important subcellular component of dinoflgaellate cells in regard to various aspects of cell surface associated ecophysiology but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella using a combination of 2-D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins were separated from A. catenella, respectively. Comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidentially identified in the 2-D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database by de novo sequence analysis and homology-based search. Majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate CWPs of dinoflagellates which provides a potential tool for future comprehensive characterization of CWPs and elucidation of physiological functions of CWPs in dinoflagellates.
keywords: Dinoflagellate Alexandrium catenella cell wall proteins fluorescence difference gel electrophoresis mass spectrometry de novo sequencing Homology searchkey
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利用二维电泳和质谱分析法鉴定有毒甲藻亚历山大藻的细胞壁蛋白
摘要:细胞壁是联系甲藻多种细胞表面生理生态学的重要亚细胞组分,但大范围的细胞壁蛋白及其功能尚未阐明。本研究利用荧光双向差异凝胶电泳(2-D DIGE)联合MALDI TOF-TOF 质谱手段鉴定和表征了有毒甲藻链状亚历山大藻的细胞壁蛋白。通过顺序提取法和热激法,细胞壁蛋白和质体蛋白依次从链状亚历山大藻中被提取和分离。比对顺次提取出的并用Cy3标记的细胞壁蛋白和用Cy5标记的质体蛋白,在2-D DIGE胶上鉴定出了120个可信的细胞壁蛋白。通过de novo 序列分析和基于同源性的搜索,这些蛋白与蛋白库中的同源蛋白成阳性匹配。虽然鉴定的多数细胞壁蛋白是未知功能或没有注释的假想或推测性蛋白,但也尝试性地鉴定到了细胞壁修饰酶,细胞壁结构蛋白,转运/结合蛋白,信号蛋白和防御蛋白,这与它们在细胞生理学中细胞外基质预想功能一致。此项工作首次探索了甲藻细胞壁蛋白,这将为今后全面阐述甲藻细胞壁蛋白特征及其生理学功能提供了潜在的工具手段。
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利用二维电泳和质谱分析法鉴定有毒甲藻亚历山大藻的细胞壁蛋白
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