Abstract
Native mass spectrometry has emerged as a powerful tool for structural biology as it enables the evaluation of molecules as they occur in their physiological conditions. Ion mobility spectrometry-mass spectrometry (IMS-MS) has shown essential in these analyses as it allows the measurement of the shape of a molecule, denoted as its collision cross section (CCS), and mass. The structural information garnered from native IMS-MS provides insight into the tertiary and quaternary structure of proteins and can be used to validate NMR or crystallographic X-ray structures. Additionally, due to the rapid nature (millisecond measurements) and ability of IMS-MS to analyze heterogeneous solutions, it can be used to address structural questions not possible with traditional structural approaches. Herein, we applied multiple solution conditions to systematically denature bovine Cu/Zn-superoxide dismutase (SOD1) and assess its unfolding pathway from the holo-dimer to the holo-monomer, single-metal monomer, and apo-monomer. Additionally, we compared and noted 1–2% agreement between CCS values from both drift tube IMS and trapped IMS for the SOD1 holo-monomer and holo-dimer. The observed CCS values were in excellent agreement with computational CCS values predicted from the homo-dimer crystal structure, showcasing the ability to use both IMS-MS platforms to provide valuable structural information for molecular modeling of protein interactions and structural assessments.
Highlights
The elucidation of protein structures is of great interest to biochemists and structural biologists, as their specific interactions and arrangements have an intimate association with their function
ion mobility spectrometry and mass spectrometry (MS) (IMS-MS) is a powerful technique for the comprehensive elucidation of protein folding and dynamics in circumstances where other biophysical methods, such as cryo-electron microscopy (cryo-EM) or crystallography, fail to detect intermediates, subtle changes, or cannot perform due to low concentrations
In its correctly folded form, superoxide dismutase 1 (SOD1) exists as a dimer with both a Zn and Cu metal ion bound to each monomer subunit (Lynch et al, 2004; Ding and Dokholyan, 2008; Kayatekin et al, 2008)
Summary
The elucidation of protein structures is of great interest to biochemists and structural biologists, as their specific interactions and arrangements have an intimate association with their function. While the conformation of proteins in their native state has traditionally been investigated using biophysical techniques such as X-ray crystallography, cryo-electron microscopy (cryo-EM), and nuclear magnetic resonance (NMR), advancements in native mass spectrometry (MS) have led to its emergence as another important tool. Native MS offers many advantages over the more traditional methods as it requires less sample compared to NMR or X-ray crystallography, is more tolerant of heterogeneous samples, and allows the analysis of protein structures that fail to take on the orderly arrangement necessary for crystallization. IMS is a gas-phase separation technique that measures the mobility of a gaseous ion through an inert buffer gas, typically nitrogen or helium, and under the influence of an electric field (Mason and Schamp, 1958). The ion mobility measurements can be used to determine the ion-neutral collision cross-section (CCS), a molecular descriptor that gives information about the size of a molecule (Mason and Schamp, 1958; Revercomb and Mason, 1975)
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