Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, United States of America.
Agile BioFoundry, Lawrence Berkeley National Laboratory, Emeryville, CA, United States of America.
PLoS One. 2022 Feb 25;17(2):e0264467. doi: 10.1371/journal.pone.0264467. eCollection 2022.
Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated liquid handler systems are increasingly used to overcome these issues for many of the sample preparation steps. Here, we detail a step-by-step protocol to prepare samples for bottom-up proteomic analysis for Gram-negative bacterial and fungal cells. The full modular protocol consists of three optimized protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of protein from 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 Pseudomonas putida and Rhodosporidium toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with protein relative quantity variance (CV%) below 15%.
手动蛋白质组学样品制备方法限制了样品通量，并且当必须分析数千个样品时，通常会导致数据质量较差。自动化液体处理系统越来越多地用于克服许多样品制备步骤中的这些问题。在这里，我们详细介绍了用于革兰氏阴性细菌和真菌细胞的自上而下的蛋白质组分析的样品制备的逐步方案。完整的模块化方案由三个优化的方案组成：（A）裂解革兰氏阴性细菌和真菌细胞; （B）定量提取的蛋白质量; （C）标准化蛋白质的量并建立胰蛋白酶消化。这些方案旨在促进数百个样品的快速，低方差样品制备，易于在广泛使用的贝克曼库尔特 Biomek 自动化液体处理机上实现，并且为未来的方案开发提供了灵活性。通过使用此工作流程，可以在不到一个小时的时间内从 96 个样品中制备 50 微克蛋白质进行胰蛋白酶消化。我们通过分析 47 个恶臭假单胞菌和红酵母样菌样品验证了这些方案，并表明这种模块化工作流程为高通量应用提供了稳健，可重复的蛋白质组学样品。这些方案的预期结果是，从革兰氏阴性细菌和真菌细胞中制备了 94 个肽样品，用于自上而下的定量蛋白质组学分析，而无需脱盐柱纯化，并且蛋白质相对量变异系数（CV％）低于 15％。
