Effects of bacterial inactivation methods on downstream proteomic analysis
Effects of bacterial inactivation methods on downstream proteomic analysis
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DOI：
10.1016/j.mimet.2015.01.015
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发表时间：
2015-05-01
影响因子：
2.2
通讯作者：
Kreuzer, Helen W.
中科院分区：
文献类型：
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作者：
Lin, Andy;Merkley, Eric D.;Kreuzer, Helen W.
为了保护研究人员并遵守当地和联邦法规，病原微生物样品的灭活通常是必要的。就其性质而言，生物灭活导致微生物样品发生变化，可能影响观察到的实验结果。虽然已经评估了灭活诱导的材料损伤，如DNA，各种灭活策略对蛋白质组学数据的影响，据我们所知，尚未讨论。为此，我们灭活鼠疫耶尔森氏菌和大肠杆菌样品高压灭菌，乙醇，或辐照处理，以确定如何灭活改变液相色谱-串联质谱数据质量以及细胞的表观蛋白质含量。不同方法灭活的样品的蛋白质组数据高度相似，Pearson相关系数分别为0.822 ~ 0.985和0.816 ~ 0.985。coil和Y. pestis，表明灭活对所鉴定的蛋白质组只有轻微的影响。此外，光谱质量指标（如各种数据库搜索算法评分的分布）在灭活方法中保持不变，表明灭活不会明显降低光谱质量。尽管灭活导致的总体变化很小，但存在可检测的趋势。例如，单侧Fischer精确检验确定，在高压灭菌灭活样品后，周质蛋白的观察丰度降低（E. coli，α = 4.97 × 10 ~（-4）;鼠疫菌）和辐射（α = 9.43 × 10 ~（-7））。coli，α = 1.21 × 10 ~（-5）。鼠疫菌）。根据我们的数据，如果样品灭活是必要的，我们建议用乙醇处理灭活，其次优先考虑辐照。(C)2015 Elsevier B. V.版权所有。
Inactivation of pathogenic microbial samples is often necessary for the protection of researchers and to comply with local and federal regulations. By its nature, biological inactivation causes changes to microbial samples, potentially affecting observed experimental results. While inactivation-induced damage to materials such as DNA has been evaluated, the effect of various inactivation strategies on proteomic data, to our knowledge, has not been discussed. To this end, we inactivated samples of Yersinia pestis and Escherichia coil by autoclave, ethanol, or irradiation treatment to determine how inactivation changes liquid chromatography-tandem mass spectrometry data quality as well as apparent protein content of cells. Proteomic datasets obtained from aliquots of samples inactivated by different methods were highly similar, with Pearson correlation coefficients ranging from 0.822 to 0.985 and 0.816 to 0.985 for E. coil and Y. pestis, respectively, suggesting that inactivation had only slight impacts on the set of proteins identified. In addition, spectral quality metrics such as distributions of various database search algorithm scores remained constant across inactivation methods, indicating that inactivation does not appreciably degrade spectral quality. Though overall changes resulting from inactivation were small, there were detectable trends. For example, one-sided Fischer exact tests determined that periplasmic proteins decrease in observed abundance after sample inactivation by autoclaving (alpha = 1.71 x 10(-2) for E. coli, alpha = 4.97 x 10(-4) for Y. pestis) and irradiation (alpha = 9.43 x 10(-7) for E. coli, alpha = 1.21 x 10(-5) for Y. pestis) when compared to controls that were not inactivated. Based on our data, if sample inactivation is necessary, we recommend inactivation with ethanol treatment with secondary preference given to irradiation. (C) 2015 Elsevier B.V. All rights reserved.