Project Summary
Advancements in biophysical techniques, such as X-ray and cryoEM, have undoubtedly accelerated
determination of protein structure. However, it still remains challenging to capture snapshots of protein
folding intermediates, including non-native states, and breathing motions that protein assemblies
undergo to perform their biological function. Moreover, understanding how molecules, such as lipids,
modulate protein structure and function is of paramount biological importance. Over the past two
decades, mass spectrometry (MS) of intact protein complexes, often referred to as native MS, has
emerged as an indispensable biophysical technique whereby non-covalent interactions and protein
structure are preserved within the mass spectrometer. Native MS is a rapid and sensitive technique
that has already provided invaluable information on subunit stoichiometry and topology, allostery and
cooperativity for individual ligand binding events, including their binding thermodynamics. The coupling
with ion mobility (IM), a separation technique based on molecule charge and shape, further enhances
the capabilities of native MS where it has enabled collision cross section (CCS) measurements for large
protein complexes, identification of different conformations for peptides and stabilizing ligands using
collision induced unfolding, and insight in folded and denatured structure(s) of proteins. However, low-
resolution commercial IM-MS instrumentation has not changed since its introduction 12 years ago.
Herein, this proposal seeks to develop transformative native IM-MS technologies with high-resolution
IM and MS capabilities that can address modern questions in structural biology, such as conformational
dynamics, including those that may have remained “hidden”, within membrane transporters under
turnover conditions. In order to achieve these transformative goals, an interdisciplinary team of
researchers whose expertise spans the fields of protein biophysics, expression and purification of
proteins inclusive of membrane proteins, as well as traditional protein structure characterization, such
as X-ray crystallography, has been assembled. Team members also possess decades of experience
in the field of mass spectrometry inclusive of fundamental ion chemistry/physics, seminal contributions
that have spawned MS proteomics, and related areas of analytical mass spectrometry and ion mobility-
mass spectrometry. Collectively, the background and expertise of this research team is uniquely
positioned to transform the field of IM-MS in the area of structural biology. In short, the proposed
transformative research will lead to forefront IM-MS instrumentation that is poised to provide
unprecedented insights into the structure and assembly of protein complexes and push the field into
new frontiers of research.