Capillary electrophoresis analysis of different variants of the amyloidogenic protein β₂‐microglobulin as a simple tool for misfolding and stability studies

Abstract

Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild-type β2 -microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25-42°C is induced inside the capillary for the two pathogenic proteins (wtβ2 -microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.

Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry

“…Unwanted interactions between proteins and negatively charged silanol groups on the inner wall of fused-silica capillaries can be prevented by applying MS-compatible coatings, allowing efficient separation of proteins in a wide range of conditions [ 26 ]. CE has shown particularly useful in revealing charge heterogeneity among protein degradation products and impurities, but also has shown the ability to separate positional isomers and conformers [ 25 , 27 , 28 ].…”

Capillary Electrophoresis: Trends and Recent Advances

“…CE allows protein analysis under near-physiological conditions, allowing detection of the different conformers. 149 − 151 For example, the unfolding of wild-type β2-microglobulin under nondenaturing conditions was investigated with CE-UV 149 and CE-ESI-MS. 150 In the latter study, several interfaces and mass spectrometers were compared. With CE-ESI-TOF MS proteoforms differing by 1 Da only could be assigned and sheathless interfacing appeared best suited to preserve protein structure integrity.…”

“…CE allows protein analysis under near-physiological conditions, allowing detection of the different conformers. − For example, the unfolding of wild-type β2-microglobulin under nondenaturing conditions was investigated with CE-UV and CE-ESI-MS . In the latter study, several interfaces and mass spectrometers were compared.…”

Native Capillary Electrophoresis–Mass Spectrometry of Near 1 MDa Non‐Covalent GroEL/GroES/Substrate Protein Complexes

“…CE‐based methods were developed to study the folding, unfolding, or misfolding of proteins. [ 17 , 18 , 19 ] Protein–ligand affinity interactions can also be probed with CE, which enables the measurement of binding stoichiometry and binding constants. [ 20 , 21 , 22 , 23 ] For instance, CE was exploited to determine the association and dissociation rates of the GroEL–GroES complex in the presence of ATP or ADP.…”