Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis.
Anal Chem
; 96(21): 8243-8248, 2024 05 28.
Article
em En
| MEDLINE
| ID: mdl-38733603
ABSTRACT
Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oxirredução
/
Dissulfetos
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article