Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes.
Evaluation of Sample Preservation and Storage Methods for Metaproteomics Analysis of Intestinal Microbiomes.
复制标题
DOI：
10.1128/spectrum.01877-21
复制
发表时间：
2021-12-22
影响因子：
3.7
通讯作者：
Kleiner M
中科院分区：
文献类型：
--
作者：
Mordant A;Kleiner M
使用元组学方法研究肠道微生物组的关键步骤是在分析前保存样品。保存对于测量基因表达的方法至关重要，例如用于识别和定量微生物组中蛋白质的元蛋白质组学。肠道微生物组样本通常通过快速冷冻和-80 °C储存来储存，但一些实验设置不允许立即冷冻样本。在这项研究中，我们评估了在不可能进行快速冷冻时保存粪便微生物组样本进行元蛋白质组学分析的方法。我们收集了C57 BL/6小鼠的粪便样本，并使用以下方法将其储存1周和4周：在液氮中快速冷冻、浸入RNAlater中、浸入95%乙醇中、浸入RNAlater样缓冲液中以及这些方法的组合。储存后，我们提取蛋白质并制备肽用于液相色谱串联质谱（LC-MS/MS）分析，以鉴定和定量肽和蛋白质。所有样品产生高度相似的元蛋白质组，除了乙醇保存的样品在蛋白质鉴定和蛋白质丰度谱方面与所有其他样品不同。快速冷冻和RNAlater（或RNAlater样处理）产生的元蛋白质组仅略有不同，不到0.7%的已鉴定蛋白质丰度不同。相比之下，乙醇保存导致平均9.5%的所鉴定的蛋白质在乙醇和其他处理之间丰度不同。我们的研究结果表明，在室温下保存在RNAlater或RNAlater样溶液中，在元蛋白质组学分析之前保存肠道微生物组样品的冷冻效果良好。元蛋白质组学是研究肠道微生物组的有力工具。通过鉴定和定量微生物组样品中大量的微生物、饮食和宿主蛋白质，元蛋白质组学为微生物群落成员的活动和功能提供了直接证据。元蛋白质组学工作流程的关键步骤是在分析前保存样品，因为蛋白质谱容易响应环境条件的变化（空气暴露，温度变化等）而快速变化。本研究评估了不同保存处理对肠道微生物组样品的元蛋白质组的影响。与先前关于保存粪便样品用于元蛋白质组学分析的工作相比，我们确保样品保存的所有步骤都是相同的，因此所有差异都可以归因于保存方法。
A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash-freezing and storage at −80°C, but some experimental setups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash-freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: flash-freezing in liquid nitrogen, immersion in RNAlater, immersion in 95% ethanol, immersion in a RNAlater-like buffer, and combinations of these methods. After storage, we extracted protein and prepared peptides for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNAlater (or RNAlater-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNAlater or an RNAlater-like solution performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses. IMPORTANCE Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members. A critical step for metaproteomics workflows is preserving samples before analysis because protein profiles are susceptible to fast changes in response to changes in environmental conditions (air exposure, temperature changes, etc.). This study evaluated the effects of different preservation treatments on the metaproteomes of intestinal microbiome samples. In contrast to prior work on preservation of fecal samples for metaproteomics analyses, we ensured that all steps of sample preservation were identical so that all differences could be attributed to the preservation method.