Shenzhen Institute of Advanced Technology Shenzhen, China (People's Republic)
Introduction: Proteins are the main bearers of life activities, and their biological functions are determined by their higher order structure. Mass spectrometry (MS) is widely used in protein analysis due to its high sensitivity and resolution, providing information on protein structure, interactions, and post-translational modifications. Unlike bottom-up mass spectrometry, which analyzes proteolysis, top-down methods are able to preserve more information about protein structure by analyzing them at the intact protein level.
Materials and
Methods: Labeling is often used to present mass differences between proteins of different structures, and then mass spectrometry is used to detect them, such as hydrogen deuterium exchange mass spectrometry (HDX-MS). This kind of sample processing prior to mass spectrometry often requires a time-consuming and labor-intensive preparation process, which makes it difficult to perform real-time analysis online, especially for the kinetic analysis of the higher-order structure of precious sample proteins. As a small and integrated device, microfluidic chip has the advantages of low consumption, automation, and online real-time detection, which provides a new scheme for mass spectrometry analysis of protein higher order structure.
Results, Conclusions, and Discussions: In this work, we demonstrate a strategy for on-line real-time top-down mass spectrometry analysis of protein higher-order structure and kinetics using microfluidic chips, which can be flexibly and easily connected to mass spectrometers, with a wide range of mixing ratios, low detection limits, fast equilibration, and real-time top-down HDX-MS. It is suitable for native top-down mass spectrometry and hydrogen/deuterium exchange mass spectrometry at temporal resolution, provides sufficient ionization efficiency for intact proteins and protein complexes, is suitable for precise control of multiple reaction flows and reaction time operations, and is compatible with large flow ranges in the submicroliters per minute. The performance of TDK-MS chips has been validated using multiple systems, such as monoclonal antibodies, antibody-antigen complexes, and coexisting protein conformational isoforms. This research strategy is conducive to the fine structure analysis and methodological development of biological macromolecules.
