High-throughput proteomics of nanogram-scale samples with Zeno SWATH DIA

The ability to conduct high-quality proteomic experiments in high throughput has opened new avenues in clinical research, drug discovery, and systems biology. Next to an increase in quantitative precision, recent developments in high-throughput proteomics have also gained proteomic depth, to the extent that earlier gaps between classic and high-throughput experiments have significantly narrowed. Here we introduce and benchmark Zeno SWATH, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) in order to increase proteomic depth and dynamic range in proteomic experiments. Combined with the high acquisition speed, these gains in sensitivity are particularly attractive for conducting high-throughput proteomics experiments with high chromatographic flow rates and fast gradients. We demonstrate that when combined with either micro-flow- or analytical-flow-rate chromatography, Zeno SWATH increases protein identification in complex samples 5- to 10-fold when compared to current SWATH acquisition methods on the same instrument. Using 20-min micro-flow chromatography, Zeno SWATH identified > 6,000 proteins from a 62.5 ng load of human cell lysate with more than 5,000 proteins consistently quantified in triplicate injections with a median CV of 6%. Using 5-min analytical-flow-rate chromatography (800 µl/min), Zeno SWATH identified 4,907 proteins from a triplicate injection of 2 µg of a human cell lysate; or more than 3,000 proteins from 250 ng tryptic digest. Zeno SWATH hence facilitates precise proteomic experiments with small sample amounts using a fast and robust high flow-rate chromatographic method, broadening the application space that requires precise proteomic experiments on a large scale.具备高通量开展高质量蛋白质组学实验的能力，为临床研究、药物研发以及系统生物学领域开辟了全新的研究路径。除定量精度的提升之外，高通量蛋白质组学的最新进展还实现了蛋白质组覆盖深度的拓展，乃至经典蛋白质组学实验与高通量实验之间原本存在的差距已大幅缩小。在此我们介绍并评测了Zeno SWATH（数据非依赖性采集技术，data-independent acquisition），该技术通过线性离子阱脉冲（Zeno阱脉冲，Zeno trap pulsing）方案，提升蛋白质组学实验中的组学覆盖深度与动态范围。结合其极高的采集速度，这种灵敏度的提升对于采用高色谱流速与快速梯度的高通量蛋白质组学实验而言极具吸引力。我们通过实验证实，在相同仪器平台上，将Zeno SWATH与微流或分析流色谱系统联用时，其对复杂样本的蛋白质鉴定数量相较于当前的SWATH采集方法提升了5至10倍。采用20分钟微流色谱方案时，Zeno SWATH可从62.5 ng的人类细胞裂解物样本中鉴定出超过6000种蛋白质，且在三次重复进样中可稳定定量超过5000种蛋白质，其变异系数（CV）中位数为6%。采用5分钟分析流色谱方案（流速800 µl/min）时，Zeno SWATH可在2 µg人类细胞裂解物的三次重复进样中鉴定出4907种蛋白质；或从250 ng的胰蛋白酶酶解产物中鉴定出超过3000种蛋白质。因此，Zeno SWATH可借助快速且稳定的高流速色谱方法，实现小样本量下的高精度蛋白质组学实验，拓展了需要开展大规模高精度蛋白质组学实验的应用领域。