Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes

Mass spectrometry-based quantitative proteomic analysis has proven valuable for clinical and biotechnology-related research and development. Improvements in sensitivity, resolution, and robustness of mass analyzers have also added value. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications where thousands of samples per month must be analyzed. To alleviate these issues, we developed a “cells-to-peptides” automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes 2 h to process 96 samples from cell pellets to the initiation of the tryptic digestion step and can process 384 samples in parallel. We measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography–mass spectrometry systems. The automated workflow was tested by preparing 96 Escherichia coli samples and quantifying over 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly multiplexed LC-MRM–MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications.基于质谱的定量蛋白质组学分析已被证实对临床与生物技术相关的研发工作具有重要价值。质量分析器的灵敏度、分辨率与稳定性的提升进一步拓展了其应用潜力。然而，手动样品制备流程往往是样品通量的瓶颈，且可能导致重现性不佳，尤其是对于每月需分析数千份样品的应用场景而言。为缓解这些问题，我们开发了一套面向革兰氏阴性菌与真菌的“从细胞到肽段”自动化工作流程，涵盖细胞裂解、蛋白质沉淀、重悬、定量、归一化及胰蛋白酶消化步骤。该工作流程可在2小时内完成96份样品从细胞沉淀到胰蛋白酶消化步骤启动前的处理，且支持384份样品的并行处理。我们针对不同生物量的细胞开展了蛋白质提取效率检测，并针对标准液相色谱-质谱联用系统优化了该流程。通过制备96份大肠杆菌样品并对超过600条肽段进行定量分析，我们对该自动化工作流程进行了验证，其结果的中位变异系数为15.8%。采用高度多重化液相色谱-多反应监测-质谱采集方法，对另外三种生物体的检测也得到了相似的技术变异度。上述结果表明，该自动化样品制备工作流程可为高通量应用提供稳定且重现性良好的蛋白质组学样品。