Aberrant protein oligomerization is a hallmark of neurodegenerative disorders, yet the conformational and kinetic underpinnings of early aggregation remain poorly understood due to the inability of structural techniques to capture transient, low-abundance oligomeric intermediates. This necessitates the development of a methodology that can characterize the conformational states related to protein unfolding and thus allow for the investigation of the molecular mechanism responsible for disease progression. Here, we demonstrate how temperature-controlled nanoelectrospray ionization (TC-nESI) combined with high-resolution ion mobility-mass spectrometry (IM-MS), surface-induced dissociation (SID), and limited proteolysis can be used to define the misfolding and oligomerization landscape of bovine Cu/Zn superoxide dismutase (SOD1). This integrative approach enables real-time detection of coexisting intermediates, and captures molecular events including metal-induced stability, monomer unfolding and assembly into heterogeneous soluble oligomers. Our results reveal that both holo- and apo-SOD1 undergo dimer dissociation followed by monomer misfolding and assembly into heterogeneous non-native oligomers, and that slow thermal ramping promotes the accumulation of misfolded monomers and higher-order complexes. Apo-SOD1 that lacks stabilizing metal cofactors, forms more compact and kinetically distinct oligomers via monomeric, dimeric and trimeric intermediates. Proteolysis and heat-induced fragmentation identify loops V, VI, VII, and the C-terminus as key labile regions contributing to oligomer interface formation, predominantly through hydrophobic interactions. Our findings establish a mechanistically rich model for early aggregation and demonstrate the capability of TC-nESI-IM-MS to temporally and structurally resolve misfolding transitions and oligomeric populations in a single experiment. This platform provides a framework to dissect oligomerization pathways relevant to neurodegenerative diseases.
Abstract
Journal Article
eng
42125835
Svingou, Despoina, et al. "Tracking Protein Misfolding and Oligomerization: a Temperature-Controlled Ion Mobility-Mass Spectrometry Approach." Analytical Chemistry, 2026.
Svingou D, McAlary L, Harrison JA, et al. Tracking Protein Misfolding and Oligomerization: A Temperature-Controlled Ion Mobility-Mass Spectrometry Approach. Anal Chem. 2026.
Svingou, D., McAlary, L., Harrison, J. A., & Zenobi, R. (2026). Tracking Protein Misfolding and Oligomerization: A Temperature-Controlled Ion Mobility-Mass Spectrometry Approach. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.5c06100
Svingou D, et al. Tracking Protein Misfolding and Oligomerization: a Temperature-Controlled Ion Mobility-Mass Spectrometry Approach. Anal Chem. 2026 May 13; PubMed PMID: 42125835.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR
T1 - Tracking Protein Misfolding and Oligomerization: A Temperature-Controlled Ion Mobility-Mass Spectrometry Approach.
AU - Svingou,Despoina,
AU - McAlary,Luke,
AU - Harrison,Julian Alexander,
AU - Zenobi,Renato,
Y1 - 2026/05/13/
PY - 2026/5/13/pubmed
PY - 2026/5/13/medline
PY - 2026/5/13/entrez
JF - Analytical chemistry
JO - Anal Chem
N2 - Aberrant protein oligomerization is a hallmark of neurodegenerative disorders, yet the conformational and kinetic underpinnings of early aggregation remain poorly understood due to the inability of structural techniques to capture transient, low-abundance oligomeric intermediates. This necessitates the development of a methodology that can characterize the conformational states related to protein unfolding and thus allow for the investigation of the molecular mechanism responsible for disease progression. Here, we demonstrate how temperature-controlled nanoelectrospray ionization (TC-nESI) combined with high-resolution ion mobility-mass spectrometry (IM-MS), surface-induced dissociation (SID), and limited proteolysis can be used to define the misfolding and oligomerization landscape of bovine Cu/Zn superoxide dismutase (SOD1). This integrative approach enables real-time detection of coexisting intermediates, and captures molecular events including metal-induced stability, monomer unfolding and assembly into heterogeneous soluble oligomers. Our results reveal that both holo- and apo-SOD1 undergo dimer dissociation followed by monomer misfolding and assembly into heterogeneous non-native oligomers, and that slow thermal ramping promotes the accumulation of misfolded monomers and higher-order complexes. Apo-SOD1 that lacks stabilizing metal cofactors, forms more compact and kinetically distinct oligomers via monomeric, dimeric and trimeric intermediates. Proteolysis and heat-induced fragmentation identify loops V, VI, VII, and the C-terminus as key labile regions contributing to oligomer interface formation, predominantly through hydrophobic interactions. Our findings establish a mechanistically rich model for early aggregation and demonstrate the capability of TC-nESI-IM-MS to temporally and structurally resolve misfolding transitions and oligomeric populations in a single experiment. This platform provides a framework to dissect oligomerization pathways relevant to neurodegenerative diseases.
SN - 1520-6882
UR - https://www.unboundmedicine.com/prime/citation/42125835/Tracking_Protein_Misfolding_and_Oligomerization:_A_Temperature-Controlled_Ion_Mobility-Mass_Spectrometry_Approach.
DB - PRIME
DP - Unbound Medicine
ER -
