Design of experiments for the optimization of sample preparation for bottom-up targeted protein LC-MS/MS workflows.

IF 1.8 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Bioanalysis Pub Date : 2025-05-01 Epub Date: 2025-06-10 DOI:10.1080/17576180.2025.2515004

Szabolcs Szarka, Margret Thorsteinsdottir, Robert Wheller

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引用次数: 0

Abstract

Aims: 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.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

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优化自下而上靶向蛋白LC-MS/MS工作流程制备样品的实验设计。

目的：实验设计（DOE）是解决复杂科学问题的一种通用而有效的方法。我们的目的是评估其在多步骤优化中的可行性，包括自下而上的样品制备，用于UPLC-MS/MS分析蛋白质。材料和方法：模型分析物为人IgG1单克隆抗体，加入大鼠血浆中，经过还原、烷基化和消化处理，用于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。结果：DOE筛选显示，尿素对替代肽反应的改善作用最大，而胍对替代肽反应的抑制作用显著。DOE优化后，DTLM、FNWY、TPEV和VVSV替代肽在短时间内的响应分别提高了2倍、10倍、10倍和50倍。结论：DOE方法成功地应用于8个变性、还原和消化参数的综合优化。结果表明，DOE是优化蛋白质样品制备的有效工具，并成功地证明了DOE模型的预测能力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

审稿时长

2 months

期刊介绍： Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing. The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality. Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing. The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques. Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.