Combining UVPD and IM-MS for structural analysis of biomolecules

  • Alina Theisen

Student thesis: Phd


Proteins are crucial for virtually all cellular processes and life; studying the way they fold into three-dimensional structures, or the lack of defined structure, and their dynamics is therefore imperative. Ion mobility mass spectrometry is ideally suited for obtaining global structural information, but requires coupling with other methods to afford local structural details. We have modified a Synapt G2-S to enable ultraviolet photodissociation, a fast and structurally-sensitive fragmentation method, of mass-selected ions either prior to or post ion mobility separation. Using 266 nm photons, we first demonstrate the capabilities of the instrument by sequencing peptides LHRH, GHRP-6 and the mini protein TrpCage. Mobility-selection allows separate UVPD spectra to be obtained for the m/z coincident Gramicidin A monomer and dimer, revealing differences in fragmentation patterns. Synchronising a single laser pulse to the start of the mobility-cycle allows discrimination between primary and secondary fragments. We also observe distinct spectra for two conformational families of 5+ melittin, indicating that UVPD at 266 nm may be conformer-dependent. We then applied our methodology to the proteins ubiquitin, cytochrome c and myoglobin using 213 nm photons. UVPD was carried out pre-ion mobility at different source conditions which altered the conformations prior to UVPD. Initial unfolding resulted in increased fragmentation yield as well as an increase in cleavage sites, which when mapped onto crystal structures revealed the sites in which initial unfolding occurred. We hypothesised that the differences in compact to extended UVPD spectra may be due to non-covalently linked fragments originating from folded precursors, and tested this by collisionally activating photoproducts post ion mobility. This revealed an increase in both yield and cleavage sites when applied to compact conformations indicating that non-covalently held fragments are indeed prevalent in UVPD of structured precursor conformations. In harsh source conditions, only cytochrome c exhibited an increase in a-type fragments, indicating that in-source activation resulted in loss of non-covalent bonding for ubiquitin and myoglobin. However, UVPD-IM-CID of compact conformations could not fully replicate the spectra obtained by UVPD of extended conformations even when secondary UVPD was accounted for; therefore it is apparent that inherent differences in the UVPD process exist between conformations. We further modified the source region of the instrument to allow LED irradiation within the sample capillary. For the photoreceptor UVR8, UV-IM-MS comparison of full-length and a truncated version revealed the presence of two distinct conformational families in the wild-type, a compact and a more extended one, due to the presence of disordered C- and N-terminal tails. Our setup allowed us to follow the receptor dynamics upon UV irradiation and postulate a role of the disordered tails in the photo-activation process.
Date of Award1 Aug 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorNicholas Lockyer (Supervisor) & Perdita Barran (Supervisor)


  • UVPD
  • Mass Spectrometry
  • Ion mobility

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