Diatoms play a critical role in the oceans' carbon and silicon cycles; however, a mechanistic understanding of the biochemical processes that contribute to their ecological success remains elusive. Completion of the Thalassiosira pseudonana genome provided 'blueprints' for the potential biochemical machinery of diatoms, but offers only a limited insight into their biology under various environmental conditions. Using high-throughput shotgun proteomics, we identified a total of 1928 proteins expressed by T. pseudonana cultured under optimal growth conditions, enabling us to analyze this diatom's primary metabolic and biosynthetic pathways. Of the proteins identified, 70% are involved in cellular metabolism, while 11% are involved in the transport of molecules. We identified all of the enzymes involved in the urea cycle, thereby describing the complete pathway to convert ammonia to urea, along with urea transporters, and the urea-degrading enzyme urease. Although metabolic exchange between these pathways remains ambiguous, their constitutive presence suggests complex intracellular nitrogen recycling. In addition, all C(4) related enzymes for carbon fixation have been identified to be in abundance, with high protein sequence coverage. Quantification of mass spectra acquisitions demonstrated that the 20 most abundant proteins included an unexpectedly high expression of clathrin, which is the primary structural protein involved in endocytic transport. This result highlights a previously overlooked mechanism for the inter- and intra-cellular transport of nutrients and macromolecules in diatoms, potentially providing a missing link to organelle communication and metabolite exchange. Our results demonstrate the power of proteomics, and lay the groundwork for future comparative proteomic studies and directed analyses of specifically expressed proteins and biochemical pathways of oceanic diatoms.

中文翻译：

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通过全细胞蛋白质组学破译硅藻生化途径

硅藻在海洋的碳和硅循环中起着至关重要的作用；然而，对促成其生态成功的生化过程的机械理解仍然难以捉摸。Thalassiosira pseudonana 基因组的完成为硅藻的潜在生化机制提供了“蓝图”，但仅提供了对其在各种环境条件下生物学的有限见解。使用高通量鸟枪蛋白质组学，我们总共鉴定了 1928 种由在最佳生长条件下培养的假假单胞菌表达的蛋白质，使我们能够分析这种硅藻的主要代谢和生物合成途径。在鉴定的蛋白质中，70% 参与细胞代谢，而 11% 参与分子运输。我们确定了所有参与尿素循环的酶，从而描述了将氨转化为尿素的完整途径，以及尿素转运蛋白和尿素降解酶尿素酶。尽管这些途径之间的代谢交换仍然不明确，但它们的组成存在表明复杂的细胞内氮循环。此外，所有用于碳固定的 C(4) 相关酶已被确定为丰富，具有高蛋白质序列覆盖率。质谱采集的量化表明，20 种最丰富的蛋白质包括网格蛋白的意外高表达，网格蛋白是参与内吞转运的主要结构蛋白。这一结果突出了先前被忽视的硅藻中营养物质和大分子的细胞间和细胞内运输机制，可能为细胞器通讯和代谢物交换提供了一个缺失的环节。我们的结果证明了蛋白质组学的力量，并为未来的蛋白质组学比较研究和定向分析海洋硅藻的特异表达蛋白质和生化途径奠定了基础。