Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using Top-Down Mass Spectrometry

Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment leve...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2022-05, Vol.94 (21), p.7520-7527
Main Authors: Li, Wen, Chaihu, Lingxiao, Jiang, Jialu, Wu, Bizhu, Zheng, Xuan, Dai, Rongrong, Tian, Ye, Huang, Yanyi, Wang, Guanbo, Men, Yongfan
Format: Article
Language:English
Subjects:
Antigens
Automation
Chemistry
Deuterium
Dynamic structural analysis
Flow velocity
Hydrogen-deuterium exchange
Interface analysis
Ionization
Mass spectrometry
Mass spectroscopy
Microfluidics
Monoclonal antibodies
Proteins
Proteomics
Scientific imaging
Semiconductors
Spatial discrimination
Spatial resolution
Spectroscopy
Structural analysis
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Summary:Characterization of protein higher-order structures and dynamics is essential for understanding the biological functions of proteins and revealing the underlying mechanisms. Top-down mass spectrometry (MS) accesses structural information at both the intact protein level and the peptide fragment level. Native top-down MS allows analysis of a protein complex’s architecture and subunits’ identity and modifications. Top-down hydrogen/deuterium exchange (HDX) MS offers high spatial resolution for conformational or binding interface analysis and enables conformer-specific characterization. A microfluidic chip can provide superior performance for front-end reactions useful for these MS workflows, such as flexibility in manipulating multiple reactant flows, integrating various functional modules, and automation. However, most microchip-MS devices are designed for bottom-up approaches or top-down proteomics. Here, we demonstrate a strategy for designing a microchip for top-down MS analysis of protein higher-order structures and dynamics. It is suitable for time-resolved native MS and HDX MS, with designs aiming for efficient ionization of intact protein complexes, flexible manipulation of multiple reactant flows, and precise control of reaction times over a broad range of flow rates on the submicroliter per minute scale. The performance of the prototype device is demonstrated by measurements of systems including monoclonal antibodies, antibody–antigen complexes, and coexisting protein conformers. This strategy may benefit elaborate structural analysis of biomacromolecules and inspire method development using the microchip-MS approach.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.2c00077