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

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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Abstract

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.