推进蛋白质异质性分析：纳米流压力动员用于精确的icIEF分离和在线质谱检测

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry Pub Date : 2025-03-02 DOI:10.1016/j.ab.2025.115825

Teresa Kwok, She Lin Chan, Niusheng Xu, Tiemin Huang, Tao Bo

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引用次数: 0

摘要

本研究解决了基于成像毛细管等电聚焦（icIEF）的工作流程中高分辨率蛋白质电荷变异分馏和高效在线质谱（MS）检测的挑战。由于扩散和不受控制的动员，icIEF经常面临效率、峰完整性和检测灵敏度的限制。为了克服这些问题，我们开发了一种新的icIEF分馏框架，该框架将纳米流压力动员与毛细管直径转换技术（CDTT）相结合。采用粒径为320 μm的分离通道和粒径为50 μm的传递毛细管，研究了影响分离效率的电泳和纳米流输运机制。评估了这些创新对电荷变形形态峰面积、高度和宽度的影响，显示出精度的提高。将这些见解应用于200 μm ID的分离通道系统，提高了分离效率和icIEF-MS灵敏度。这项研究为生物制药开发和监管应用中的电荷异质性分析提供了一个可扩展的、高精度的解决方案。

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$Advancing protein heterogeneity analysis: Nano-flow pressure mobilization for precise icIEF fractionation and online MS detection$

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Advancing protein heterogeneity analysis: Nano-flow pressure mobilization for precise icIEF fractionation and online MS detection

This study addresses the challenges of high-resolution protein charge variant fractionation and efficient online mass spectrometry (MS) detection in imaged capillary isoelectric focusing (icIEF)-based workflows. icIEF often faces limitations in efficiency, peak integrity, and detection sensitivity due to diffusion and uncontrolled mobilization. To overcome these, we developed a novel icIEF fractionation framework that integrates nano-flow pressure mobilization with the capillary diameter transformation technique (CDTT). Using a model system with a 320 μm ID separation channel and a 50 μm ID transfer capillary, we investigated the electrophoretic and nano-flow transport mechanisms influencing fractionation efficiency. The impact of these innovations on peak area, height, and width for charge proteoforms was assessed, showing improvements in precision. These insights were applied to a 200 μm ID separation channel system, resulting in enhanced separation efficiency and icIEF-MS sensitivity. This study offers a scalable, high-precision solution for charge heterogeneity analysis in biopharmaceutical development and regulatory applications.

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.