Modern mass spectrometers routinely allow deep proteome coverage in a single experiment. These methods are typically operated at nano and micro flow regimes, but they often lack throughput and chromatographic robustness, which is critical for large-scale studies. In this context, we have developed, optimized and benchmarked LC-MS methods combining the robustness and throughput of analytical flow chromatography with the added sensitivity provided by the Zeno trap across a wide range of cynomolgus monkey and human matrices of interest for toxicological studies and clinical biomarker discovery, respectively. SWATH data independent acquisition (DIA) experiments with Zeno trap activated (Zeno SWATH DIA) provided a clear advantage over conventional SWATH DIA in all sample types tested with improved sensitivity, quantitative robustness and signal linearity as well as increased protein coverage by up to 9-fold. Using a 10-min gradient chromatography, up to 3,300 proteins were identified in tissues at 2 μg peptide load. Importantly, the performance gains with Zeno SWATH translated into better biological pathway representation and improved the ability to identify dysregulated proteins and pathways associated with two metabolic diseases in human plasma. Finally, we demonstrate that this method is highly stable over time with the acquisition of reliable data over the injection of 1,000+ samples (14.2 days of uninterrupted acquisition) without the need for human intervention or normalization. Altogether, Zeno SWATH DIA methodology allows fast, sensitive and robust proteomic workflows using analytical flow and is amenable to large-scale studies, especially in clinical settings. This work provides detailed method performance assessment on a variety of relevant biological matrices and serves as a valuable resource for the proteomics community.

中文翻译：

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使用 Zeno SWATH 数据独立采集对食蟹猴和人类基质进行稳健且高通量的分析流蛋白质组学分析

现代质谱仪通常允许在单个实验中进行深度蛋白质组覆盖。这些方法通常在纳流和微流条件下运行，但它们通常缺乏通量和色谱稳健性，而这对于大规模研究至关重要。在此背景下，我们开发、优化和基准化了 LC-MS 方法，将分析流色谱的稳健性和通量与 Zeno trap 提供的更高灵敏度相结合，适用于广泛的食蟹猴和人体毒理学研究和临床感兴趣的基质生物标志物发现，分别。激活 Zeno trap 的 SWATH 数据独立采集 (DIA) 实验 (Zeno SWATH DIA) 在所有测试的样品类型中提供了明显优于传统 SWATH DIA 的优势，灵敏度更高，定量稳定性和信号线性度以及高达 9 倍的蛋白质覆盖率。使用 10 分钟的梯度色谱法，在 2 μg 肽载量下，在组织中鉴定了多达 3,300 种蛋白质。重要的是，Zeno SWATH 的性能提升转化为更好的生物通路表示，并提高了识别与人类血浆中两种代谢疾病相关的失调蛋白质和通路的能力。最后，我们证明了这种方法随着时间的推移高度稳定，通过注入 1,000 多个样本（14.2 天的不间断采集）采集可靠数据，无需人工干预或标准化。总而言之，Zeno SWATH DIA 方法允许使用分析流程进行快速、灵敏和稳健的蛋白质组学工作流程，并且适用于大规模研究，特别是在临床环境中。这项工作提供了对各种相关生物基质的详细方法性能评估，并作为蛋白质组学界的宝贵资源。