Szarka Szabolcs, Thorsteinsdottir Margret, Wheller Robert
Resolian, Bioanalytics, Fordham, Cambridgeshire, UK.
Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.
Bioanalysis. 2025 May;17(10):641-650. doi: 10.1080/17576180.2025.2515004. Epub 2025 Jun 10.
Design of experiments (DOE) is a versatile and efficient approach to tackle complex scientific problems. We aimed to assess its feasibility in the optimization of the multistep, involved bottom-up sample preparation for the UPLC-MS/MS analysis of proteins.
The model analyte was a human IgG1 monoclonal antibody which was spiked into rat plasma and processed further by reduction, alkylation, and digestion for the subsequent UPLC-MS/MS analysis. The Modde Go software was used for the generation of experimental designs and for processing, analyzing and the interpretation of the data.
DOE screening revealed that urea made the biggest improvement on the surrogate peptide responses, while guanidine significantly suppressed them. DOE optimization resulted in a 2-, 10-, 10- and 50-fold response increase for the respective DTLM, FNWY, TPEV and VVSV surrogate peptide even after a short, <3-h sample preparation, when compared to a legacy method that required 2-day preparation.
The DOE approach was applied successfully for the comprehensive optimization of eight denaturation, reduction and digestion parameters. DOE was found to be an efficient tool for protein sample preparation optimization, and the predictive power of the DOE models was successfully demonstrated.
实验设计（DOE）是解决复杂科学问题的一种通用且高效的方法。我们旨在评估其在优化用于蛋白质超高效液相色谱-串联质谱（UPLC-MS/MS）分析的多步骤、复杂的自下而上样品制备过程中的可行性。
模型分析物为人IgG1单克隆抗体，将其添加到大鼠血浆中，然后通过还原、烷基化和消化进一步处理，用于后续的UPLC-MS/MS分析。使用Modde Go软件生成实验设计，并对数据进行处理、分析和解释。
DOE筛选显示，尿素对替代肽响应的改善最大，而胍则显著抑制了它们。与需要2天制备的传统方法相比，即使经过短至<3小时的样品制备，DOE优化后，相应的DTLM、FNWY、TPEV和VVSV替代肽的响应分别增加了2倍、10倍、10倍和50倍。
DOE方法成功应用于八个变性、还原和消化参数的全面优化。发现DOE是蛋白质样品制备优化的有效工具，并成功证明了DOE模型的预测能力。
