Wang Angela, Fekete Emily E F, Creskey Marybeth, Cheng Kai, Ning Zhibin, Pfeifle Annabelle, Li Xuguang, Figeys Daniel, Zhang Xu
Regulatory Research Division, Biologic and Radiopharmaceutical Drugs Directorate, Health Products and Food Branch, Health Canada, Ottawa K1A 0K9, Ontario, Canada.
Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa K1H 8M5, Ontario, Canada.
Microbiome Res Rep. 2024 Jul 3;3(3):39. doi: 10.20517/mrr.2024.21. eCollection 2024.
This study aims to evaluate the impact of experimental workflow on fecal metaproteomic observations, including the recovery of small and antimicrobial proteins often overlooked in metaproteomic studies. The overarching goal is to provide guidance for optimized metaproteomic experimental design, considering the emerging significance of the gut microbiome in human health, disease, and therapeutic interventions. Mouse feces were utilized as the experimental model. Fecal sample pre-processing methods (differential centrifugation and non-differential centrifugation), protein digestion techniques (in-solution and filter-aided), data acquisition modes (data-dependent and data-independent, or DDA and DIA) when combined with parallel accumulation-serial fragmentation (PASEF), and different bioinformatic workflows were assessed. We showed that, in DIA-PASEF metaproteomics, the library-free search using protein sequence database generated from DDA-PASEF data achieved better identifications than using the generated spectral library. Compared to DDA, DIA-PASEF identified more microbial peptides, quantified more proteins with fewer missing values, and recovered more small antimicrobial proteins. We did not observe any obvious impacts of protein digestion methods on both taxonomic and functional profiles. However, differential centrifugation decreased the recovery of small and antimicrobial proteins, biased the taxonomic observation with a marked overestimation of species, and altered the measured functional compositions of metaproteome. This study underscores the critical impact of experimental choices on metaproteomic outcomes and sheds light on the potential biases introduced at different stages of the workflow. The comprehensive methodological comparisons serve as a valuable guide for researchers aiming to enhance the accuracy and completeness of metaproteomic analyses.
本研究旨在评估实验工作流程对粪便宏蛋白质组学观察结果的影响，包括在宏蛋白质组学研究中经常被忽视的小分子和抗菌蛋白的回收率。总体目标是为优化宏蛋白质组学实验设计提供指导，考虑到肠道微生物群在人类健康、疾病和治疗干预中日益重要的意义。小鼠粪便被用作实验模型。评估了粪便样本预处理方法（差速离心和非差速离心）、蛋白质消化技术（溶液内消化和滤膜辅助消化）、与平行累积连续碎裂（PASEF）相结合的数据采集模式（数据依赖和数据独立，即DDA和DIA）以及不同的生物信息学工作流程。我们表明，在DIA-PASEF宏蛋白质组学中，使用从DDA-PASEF数据生成的蛋白质序列数据库进行无谱库搜索比使用生成的谱库能实现更好的鉴定。与DDA相比，DIA-PASEF鉴定出更多的微生物肽，定量了更多缺失值较少的蛋白质，并回收了更多的小分子抗菌蛋白。我们没有观察到蛋白质消化方法对分类学和功能谱有任何明显影响。然而，差速离心降低了小分子和抗菌蛋白的回收率，使分类学观察产生偏差，显著高估了物种，并改变了宏蛋白质组的测量功能组成。本研究强调了实验选择对宏蛋白质组学结果的关键影响，并揭示了工作流程不同阶段引入的潜在偏差。全面的方法学比较为旨在提高宏蛋白质组学分析准确性和完整性的研究人员提供了有价值的指导。
