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首页> 美国卫生研究院文献>Frontiers in Microbiology >Examination of Microbial Proteome Preservation Techniques Applicable to Autonomous Environmental Sample Collection
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Examination of Microbial Proteome Preservation Techniques Applicable to Autonomous Environmental Sample Collection

机译：适用于自主环境样品采集的微生物蛋白质组保存技术的检验

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Improvements in temporal and spatial sampling frequency have the potential to open new windows into the understanding of marine microbial dynamics. In recent years, efforts have been made to allow automated samplers to collect microbial biomass for DNA/RNA analyses from moored observatories and autonomous underwater vehicles. Measurements of microbial proteins are also of significant interest given their biogeochemical importance as enzymes that catalyze reactions and transporters that interface with the environment. We examined the influence of five preservatives solutions (SDS-extraction buffer, ethanol, trichloroacetic acid, B-PER, and RNAlater) on the proteome integrity of the marine cyanobacterium Synechococcus WH8102 after 4 weeks of storage at room temperature. Four approaches were used to assess degradation: total protein recovery, band integrity on an SDS detergent polyacrylamide electrophoresis (SDS-PAGE) gel, and number of protein identifications and relative abundances by 1-dimensional LC–MS/MS proteomic analyses. Total protein recoveries from the preserved samples were lower than the frozen control due to processing losses, which could be corrected for with internal standardization. The trichloroacetic acid preserved sample showed significant loss of protein band integrity on the SDS-PAGE gel. The RNAlater preserved sample showed the highest number of protein identifications (103% relative to the control; 520 ± 31 identifications in RNAlater versus 504 ± 4 in the control), equivalent to the frozen control. Relative abundances of individual proteins in the RNAlater treatment were quite similar to that of the frozen control (average ratio of 1.01 ± 0.27 for the 50 most abundant proteins), while the SDS-extraction buffer, ethanol, and B-PER all showed significant decreases in both number of identifications and relative abundances of individual proteins. Based on these findings, RNAlater was an effective proteome preservative, although further study is warranted on additional marine microbes.
机译：时间和空间采样频率的提高有可能为了解海洋微生物动力学打开新的窗口。近年来，人们一直在努力使自动化的采样器能够从停泊的观测站和水下自动航行器收集微生物生物量用于DNA / RNA分析。考虑到微生物蛋白质作为催化与环境接触的反应和转运蛋白的酶的生物地球化学重要性，对微生物蛋白质的测量也引起了极大的兴趣。我们研究了五种防腐剂溶液（SDS提取缓冲液，乙醇，三氯乙酸，B-PER和RNAlater）在室温下保存4周后对海洋蓝藻Synechococcus WH8102蛋白质组完整性的影响。四种方法用于评估降解：总蛋白回收率，SDS去污剂聚丙烯酰胺电泳（SDS-PAGE）凝胶上的条带完整性以及通过一维LC-MS / MS蛋白质组学分析确定的蛋白质数量和相对丰度。由于加工损失，从保存的样品中回收的总蛋白质低于冷冻对照，可以通过内部标准化对其进行校正。保存的三氯乙酸样品在SDS-PAGE凝胶上显示出明显的蛋白质条带完整性损失。 RNAlater保存的样品显示出最高的蛋白质鉴定数量（相对于对照为103％； RNAlater中520±31鉴定，而对照中的504±4），相当于冷冻对照。 RNAlater处理中单个蛋白质的相对丰度与冷冻对照的相对丰度非常相似（50个最丰富的蛋白质的平均比率为1.01）±0.27），而SDS提取缓冲液，乙醇和B-PER均显示出显着降低在单个蛋白质的鉴定数量和相对丰度方面。基于这些发现，RNAlater是一种有效的蛋白质组防腐剂，尽管有必要对其他海洋微生物进行进一步研究。

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