کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1165020 1491061 2013 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography–mass spectrometry
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Separation and characterisation of beta2-microglobulin folding conformers by ion-exchange liquid chromatography and ion-exchange liquid chromatography–mass spectrometry
چکیده انگلیسی

In this work we present for the first time the use of ion-exchange liquid chromatography to separate the native form and a partially structured intermediate of the folding of the amyloidogenic protein beta2-microglobulin. Using a strong anion-exchange column that accounts for the differences in charge exposure of the two conformers, a LC–UV method is initially optimised in terms of mobile phase pH, composition and temperature. The preferred mobile phase conditions that afford useful information were found to be 35 mM ammonium formate, pH 7.4 at 25 °C. The dynamic equilibrium of the two species is demonstrated upon increasing the concentration of acetonitrile in the protein sample. Then, the chromatographic method is transferred to MS detection and the respective charge state distributions of the separated conformers are identified. The LC–MS results demonstrate that one of the conformers is partially unfolded, compared with the native and more compact species. The correspondence with previous results obtained in free solution by capillary electrophoresis suggest that strong ion exchange LC–MS does not alter beta2-microglobulin conformation and maintains the dynamic equilibrium already observed between the native protein and its folding intermediate.

Figure optionsDownload as PowerPoint slideHighlights
► We present LC–UV and LC–MS methods to separate protein conformations.
► We demonstrate that protein conformations are at dynamic equilibrium.
► Strong anion exchange chromatography–ESI-MS does not alter β2-microglobulin conformation.
► Charge state distributions characterise the folding state of the separated species.
► The separated species can be molecular targets of drug-like molecules.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytica Chimica Acta - Volume 771, 10 April 2013, Pages 108–114
نویسندگان
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