Probing the Structures of Large Intact Biomolecules Using Mass Spectrometry

Abstract

The analysis of large intact biomolecules via mass spectrometry (MS) has developed into an invaluable technique that can provide structural information about a biomolecule’s native state. Instrumental developments in conventional MS, where a measurement of an ion’s m/z value is recorded, have increased the mass range of modern instrumentation into the 10^5-10^7 Da mass range. Charge detection MS (CDMS) is a promising alternative to conventional MS in the analysis of large intact biomolecules. CDMS takes independent measurements of the ion’s charge (z) and m/z. To date, CDMS is not a commercially available platform, which contrasts with the numerous conventional MS platforms available. In this thesis the design process of our home built CDMS instrument is detailed. Via ion trajectory simulations an ion funnel, dual hemispherical deflector analyser (HDA) and ion trap were characterised and designed. The ion funnel design was shown to achieve high transmission efficiency over a wide mass range, which will improve our instruments sensitivity for high mass ions. Furthermore, ion trajectories through the dual HDA were optimised via simulations to achieve an energy resolution of 3.25 %, which will improve the resolution of our CDMS instrument. Finally, conventional MS techniques were utilised to investigate two large protein complexes. The tetracosameric E. coli bacterioferritin (EcBfr) is in equilibrium with its dimeric form. In this work a previously unidentified octadecameric intermediate has been characterised. The flexible transcription factor IIIC (TFIIIC) complex plays an important role in the transcription of non-coding RNAs. Due to the complex’s flexibility, information on its native structures is limited. Here, extended and compact conformations of Apo-TFIIIC and DNA bound TFIIIC have been identified. It appears that the τ91 subunit is responsible for extended forms of Apo-TFIIIC and DNA bound TFIIIC.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Perdita Barran (Supervisor)