Metaproteomics可以提供有关人类肠道微生物群的功能的有价值的洞见，但由于GM的极度复杂性和异质性而具有挑战性。数据无关采集（DIA）质谱（MS）已成为传统蛋白质组学中的新兴定量技术，但在代谢蛋白质组学领域仍处于早期阶段。在此，我们应用了基于无库直接DIA（directDIA）的代谢蛋白质组学，并将directDIA与其他基于MS的代谢蛋白质组学定量技术进行了比较，以模拟微生物群中的微生物社区和粪便样品中含有已知比例的细菌，并展示了直接DIA在识别中的蛋白质组覆盖率以及定量中的准确性和精度方面的出色性能。我们对胰腺癌（PC）和轻度认知障碍（MCI）的两组临床粪便样品中的人类GM进行了表征。每个队列中约有70,000个微生物蛋白质被定量，并注释以对不同疾病中GM的分类学和功能特征进行概述。我们的工作证明了直接DIA在定量代谢蛋白质组学中的实用性，用于研究肠道微生物群及其相关的疾病发病机理。©2023.作者。

Metaproteomics can provide valuable insights into the functions of human gut microbiota (GM), but is challenging due to the extreme complexity and heterogeneity of GM. Data-independent acquisition (DIA) mass spectrometry (MS) has been an emerging quantitative technique in conventional proteomics, but is still at the early stage of development in the field of metaproteomics. Herein, we applied library-free DIA (directDIA)-based metaproteomics and compared the directDIA with other MS-based quantification techniques for metaproteomics on simulated microbial communities and feces samples spiked with bacteria with known ratios, demonstrating the superior performance of directDIA by a comprehensive consideration of proteome coverage in identification as well as accuracy and precision in quantification. We characterized human GM in two cohorts of clinical fecal samples of pancreatic cancer (PC) and mild cognitive impairment (MCI). About 70,000 microbial proteins were quantified in each cohort and annotated to profile the taxonomic and functional characteristics of GM in different diseases. Our work demonstrated the utility of directDIA in quantitative metaproteomics for investigating intestinal microbiota and its related disease pathogenesis.© 2023. The Author(s).