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MnESI-MS platform for LC-MS analysis of native protein complexes


To establish a Microflow-nanospray ESI-MS (MnESI-MS) platform for LC-native MS analysis of labile native protein complexes, that achieves high throughput, high sensitivity, and robustness, using the Newomics® MnESI source and M3 multinozzle emitters interfaced with the Thermo Scientic™ Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.


Native mass spectrometry (MS) maintains a biomolecule’s natural folded state and associated non-covalent interactions for mass spectrometry analysis, and is a powerful technique for studying the structure of intact proteins, large protein complexes, and protein-protein, protein-ligand interactions [1-4]. Currently, one of the biggest challenges for native MS is the analysis of large native protein complexes and their mixtures in a high-throughput manner. The current static nanoelectrospray MS (nanoESI-MS) method requires delicate technique for sample introduction and lengthy buffer exchange process for sample preparation, suffers from low reproducibility and robustness, and has a low sample throughput [5-8]. On the other hand, the conventional analytical flow LC-MS method has not been adopted widely for native MS studies of large bioorganic complexes because it does not have the sensitivity of static nanoESI-MS or may not be able to maintain the native state of labile protein complexes during mass spectrometry analysis. To directly address the challenges, we have developed a new Microflow-nanospray ESI-native MS (MnESI-MS) platform with a MnESI source and M3 emitters interfaced with the Q Exactive UHMR mass spectrometer, to achieve high-sensitivity and high-throughput LC-MS analysis of bioorganic complexes while maintaining their native state [9-12]. We have validated our platform using GroEL, r20S, and t20S proteasome as model complexes and demonstrated its utilities for cryo-EM sample screening.

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