Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
Unit of Microbiology and Virology, University Hospital of Sassari, Sassari, Italy.
mSystems. 2024 Jul 23;9(7):e0066124. doi: 10.1128/msystems.00661-24. Epub 2024 Jun 27.
The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although high-throughput protein cleanup and digestion methods are increasingly used in shotgun proteomics, no studies have yet critically compared such protocols using human fecal samples. In this study, human fecal protein extracts were processed using several different protocols based on three main approaches: filter-aided sample preparation (FASP), solid-phase-enhanced sample preparation (SP3), and suspension trapping (S-Trap). These protocols were applied in both low-throughput (i.e., microtube-based) and high-throughput (i.e., microplate-based) formats, and the final peptide mixtures were analyzed by liquid chromatography coupled to high-resolution tandem mass spectrometry. The FASP-based methods and the combination of SP3 with in-StageTips (iST) yielded the best results in terms of the number of peptides identified through a database search against gut microbiome and human sequences. The efficiency of protein digestion, the ability to preserve hydrophobic peptides and high molecular weight proteins, and the reproducibility of the methods were also evaluated for the different protocols. Other relevant variables, including interindividual variability of stool, duration of protocols, and total costs, were considered and discussed. In conclusion, the data presented here can significantly contribute to the optimization and standardization of sample preparation protocols in human fecal metaproteomics. Furthermore, the promising results obtained with the high-throughput methods are expected to encourage the development of automated workflows and their application to large-scale gut microbiome studies.IMPORTANCEFecal metaproteomics is an experimental approach that allows the investigation of gut microbial functions, which are involved in many different physiological and pathological processes. Standardization and automation of sample preparation protocols in fecal metaproteomics are essential for its application in large-scale studies. Here, we comparatively evaluated different methods, available also in a high-throughput format, enabling two key steps of the metaproteomics analytical workflow (namely, protein cleanup and digestion). The results of our study provide critical information that may be useful for the optimization of metaproteomics experimental pipelines and their implementation in laboratory automation systems.
粪便宏蛋白质组学在大规模肠道微生物组研究中的应用需要高通量分析和标准化的实验方案。尽管在鸟枪法蛋白质组学中越来越多地使用高通量蛋白质纯化和消化方法，但迄今为止，还没有研究对使用人类粪便样本的此类方案进行严格比较。在这项研究中，使用基于三种主要方法的几种不同方案处理人类粪便蛋白质提取物：滤过辅助样品制备（FASP）、固相增强样品制备（SP3）和悬浮捕获（S-Trap）。这些方案分别在低通量（即基于微量管）和高通量（即基于微孔板）格式中应用，并通过液相色谱与高分辨率串联质谱联用分析最终的肽混合物。基于 FASP 的方法和 SP3 与 In-StageTips（iST）的组合在通过数据库搜索针对肠道微生物组和人类序列鉴定的肽数量方面产生了最佳结果。还评估了不同方案的蛋白质消化效率、保留疏水性肽和高分子量蛋白质的能力以及方法的重现性。还考虑并讨论了其他相关变量，包括粪便的个体间变异性、方案的持续时间和总费用。总之，这里提供的数据将极大地有助于优化和标准化人类粪便宏蛋白质组学中的样品制备方案。此外，高通量方法获得的有希望的结果有望鼓励自动化工作流程的开发及其在大规模肠道微生物组研究中的应用。
重要性粪便宏蛋白质组学是一种实验方法，可用于研究肠道微生物功能，这些功能涉及许多不同的生理和病理过程。粪便宏蛋白质组学中样品制备方案的标准化和自动化对于其在大规模研究中的应用至关重要。在这里，我们比较评估了不同的方法，这些方法也可采用高通量格式，从而实现宏蛋白质组学分析工作流程的两个关键步骤（即蛋白质纯化和消化）。我们研究的结果提供了重要信息，这些信息可能有助于优化宏蛋白质组学实验流程及其在实验室自动化系统中的实施。
