Mass Spectrometry-Based Structural Proteomics for Metal Ion/Protein Binding Studies

Abstract

Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

“…Variable stoichiometry combinations of hetero‐dodecamers from 12:0 to 0:12 were identified by nMS, SID for each individual hetero‐dodecamers further revealed the lateral symmetry in the subunit arrangement, greatly improve the understanding of catalytic mechanism of PDX heterocomplexes and further explain the correlation between the catalytic activity with PDX1.2‐PDX1.3 stoichiometry (Novikova et al, 2021). Additionally, Lin and co‐workers (Lin & Gross, 2022) recently found that the sialyation, especially Neu5Gc, can stabilize transferrin conformation and inhibit its dimerization, furtherly improving its association with iron and Aβ, ultimately resulting in the transferrin‐mediated Aβ‐iron cellular co‐transporting.…”

Hydrogen/Deuterium Exchange Mass Spectrometry Provides Insights into the Role of Drosophila Testis-Specific Myosin VI Light Chain AndroCaM

“…For more discussion of this point, we cite a recent review of our efforts to understand metal-ion interactions by MS-based footprinting and HDX. 35 ■ EXPERIMENTAL PROCEDURES Materials. All chemicals, proteases, and solvents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.…”

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches

“…These techniques induce fragmentation of the peptide without major scrambling of hydrogen and deuterium, permitting the location of deuterium to be accurately determined. 15 A recent study by the FDA 16 monitored, using HDX-MS, changes in solvent accessibilities of stressed mAbs relative to an unstressed control incubated at 25 ± 2 °C, 60 ± 5% relative humidity (RH) for 6 months. Although this study provided a broad survey across several antibodies, there are still challenges to improving the spatial resolution (i.e., peptide length or number of unique peptides) and to employing robust statistical analysis for accurate and reliable measurements of subtle structural changes by using HDX-MS methods.…”

“…Tandem MS (MS/MS) fragmentation such as electron transfer dissociation, electron capture dissociation, and ultraviolet photon dissociation may also be adopted. These techniques induce fragmentation of the peptide without major scrambling of hydrogen and deuterium, permitting the location of deuterium to be accurately determined . A recent study by the FDA monitored, using HDX-MS, changes in solvent accessibilities of stressed mAbs relative to an unstressed control incubated at 25 ± 2 °C, 60 ± 5% relative humidity (RH) for 6 months.…”