Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942
Proteomic assessment of an established technique for carboxysome enrichment from Synechococcus PCC7942
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DOI：
10.1139/b05-058
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发表时间：
2005-07-01
期刊：
影响因子：
--
通讯作者：
Badger, MR
中科院分区：
文献类型：
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作者：
Long, BM;Price, GD;Badger, MR
羧基体是蓝藻中与蛋白质结合的多面体微体，含有光合作用CO2固定的关键酶-核酮糖-1，5-二磷酸羧化酶-加氧酶（Rubisco）。蓝藻基因组测序表明，蓝藻拥有一个或其他两种类型的羧基体。含有1A型Rubisco的蓝细菌具有α-羧基体，而含有1B型Rubisco的蓝细菌具有β-羧基体。由于核心的重要性，carboxysomes在蓝藻的CO2浓缩机制，了解这些结构的性质和组成是相当重要的。在努力开发技术的β-carboxysomes的结构的表征，特别是外部的蛋白质壳，我们已经进行了蛋白质组学评估的Percoll-镁2 + carboxysome富集技术，使用淡水蓝藻聚球藻属。PCC 7942。采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和多维蛋白质鉴定技术（MuDPIT）两种方法测定一种新的富含羧基组分的蛋白质含量。使用MALDI-TOF MS从富集的羧基体制备物中鉴定了总共17种蛋白质，而使用MuDPIT分析在相同材料上鉴定了122种蛋白质。通过MALDI-TOF MS将羧基体蛋白CcmM鉴定为38和58 kDa的两种不同的蛋白。唯一的其他carboxysomal蛋白鉴定的Rubisco（RbcL和RbcS）的大亚基和小亚基。缺乏证据的carboxysomal蛋白和未来的方向进行了讨论的预期充分补充的原因。
Carboxysomes are protein-bound, polyhedral microbodies within cyanobacteria, containing the key enzyme for photosynthetic CO2 fixation, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). Sequencing of cyanobacterial genomes has revealed that cyanobacteria possess one or other of two types of carboxysomes. Cyanobacteria containing form 1A Rubisco possess alpha-carboxysomes, while those with form 1B Rubisco possess beta-carboxysomes. Given the central importance of carboxysomes in the CO2-concentrating mechanism of cyanobacteria, understanding the nature and composition of these structures is of considerable importance. In an effort to develop techniques for the characterization of the structure of beta-carboxysomes, particularly the outer protein shell, we have undertaken a proteomic assessment of the Percoll-Mg2+ carboxysome enrichment technique using the freshwater cyanobacterium Synechococcus sp. PCC7942. Both matrix-assisted laser desorption-ionization - time of flight mass spectrometry (MALDI-TOF MS) and multidimensional protein identification technology (MuDPIT) methods were used to determine the protein content of a novel carboxysome-rich fraction. A total of 17 proteins were identified using MALDI-TOF MS from enriched carboxysome preparations, while 122 proteins were identified using MuDPIT analysis on the same material. The carboxysomal protein CcmM was identified by MALDI-TOF MS as two distinct proteins of 38 and 58 kDa. The only other carboxysomal proteins identified were the large and small subunits of Rubisco (RbcL and RbcS). Reasons for the lack of evidence for the expected full complement of carboxysomal proteins and future directions are discussed.