Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins
Polydispersity presents a considerable challenge for the detailed molecular characterisation of many proteins. This is because in most biophysical and structural biology approaches the molecules in solution are ensemble-averaged, obscuring differences between individual proteins or conformational st...
| Main Authors: | , , , , |
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| Format: | Journal article |
| Language: | English |
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Elsevier
2015
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| _version_ | 1797088167525875712 |
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| author | Shepherd, D Marty, M Giles, K Baldwin, A Benesch, J |
| author_facet | Shepherd, D Marty, M Giles, K Baldwin, A Benesch, J |
| author_sort | Shepherd, D |
| collection | OXFORD |
| description | Polydispersity presents a considerable challenge for the detailed molecular characterisation of many proteins. This is because in most biophysical and structural biology approaches the molecules in solution are ensemble-averaged, obscuring differences between individual proteins or conformational states. Mass spectrometry is however inherently dispersive, allowing the specific interrogation of molecules with distinct mass-to-charge ratios. Here, we exploit this intrinsic benefit to develop a means for determining directly the stoichiometries and sizes of oligomers comprising a polydisperse protein ensemble. Our method exploits the quadrupole-(ion-mobility)-(time-of-flight) geometry by submitting selected mass-to-charge ranges for ion mobility separation followed by collision-induced dissociation. In this sequential experiment the ion mobility information of the precursors is reported by the arrival times of the fragments, which are highly separated in mass-to-charge by virtue of the dissociation process. We observe small differences in the measured arrival time between fragments arising due to ion transit conditions after the ion mobility cell. To accommodate these systematic deviations, we develop a mass-to-charge dependent correction, leading to a reduction in the error of the collision cross-section measurement to around 0.5%. We characterise our method using HSP16.9, a small heat-shock protein that undergoes a mono- to polydisperse transition upon lowering pH, and reveal that the oligomers it forms have collisional cross-sections consistent with the polyhedral and double-ring architectures exhibited by other members of the protein family. |
| first_indexed | 2024-03-07T02:46:05Z |
| format | Journal article |
| id | oxford-uuid:ac1fa881-4748-44d6-8241-8c5d874435c6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T02:46:05Z |
| publishDate | 2015 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | oxford-uuid:ac1fa881-4748-44d6-8241-8c5d874435c62022-03-27T03:26:36ZCombining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteinsJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:ac1fa881-4748-44d6-8241-8c5d874435c6EnglishSymplectic Elements at OxfordElsevier2015Shepherd, DMarty, MGiles, KBaldwin, ABenesch, JPolydispersity presents a considerable challenge for the detailed molecular characterisation of many proteins. This is because in most biophysical and structural biology approaches the molecules in solution are ensemble-averaged, obscuring differences between individual proteins or conformational states. Mass spectrometry is however inherently dispersive, allowing the specific interrogation of molecules with distinct mass-to-charge ratios. Here, we exploit this intrinsic benefit to develop a means for determining directly the stoichiometries and sizes of oligomers comprising a polydisperse protein ensemble. Our method exploits the quadrupole-(ion-mobility)-(time-of-flight) geometry by submitting selected mass-to-charge ranges for ion mobility separation followed by collision-induced dissociation. In this sequential experiment the ion mobility information of the precursors is reported by the arrival times of the fragments, which are highly separated in mass-to-charge by virtue of the dissociation process. We observe small differences in the measured arrival time between fragments arising due to ion transit conditions after the ion mobility cell. To accommodate these systematic deviations, we develop a mass-to-charge dependent correction, leading to a reduction in the error of the collision cross-section measurement to around 0.5%. We characterise our method using HSP16.9, a small heat-shock protein that undergoes a mono- to polydisperse transition upon lowering pH, and reveal that the oligomers it forms have collisional cross-sections consistent with the polyhedral and double-ring architectures exhibited by other members of the protein family. |
| spellingShingle | Shepherd, D Marty, M Giles, K Baldwin, A Benesch, J Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title | Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title_full | Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title_fullStr | Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title_full_unstemmed | Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title_short | Combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
| title_sort | combining tandem mass spectrometry with ion mobility separation to determine the architecture of polydisperse proteins |
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