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(BBSRC DTP CASE) Native Mass Spectrometry approaches for Electron and Photon mediated Top Down Sequencing of Biotherapeutics


Project Description

Top down mass spectrometry, where the intact protein is introduced into the mass spectrometer and analysed as intact as well as after dissociation, offers several advantages over the more common bottom up approach, where the protein is digested into its constituent peptides before mass spectrometric analysis. The top down approach doesn’t rely on enzymatic digestion and therefore is not limited by the efficiency of this step. Additionally, the presence of several post-translational modification on the same protein isoform can be elucidated, thus giving insights into their combinatorial effects and advancing the understanding of the biological properties of the product. Fragmentation of protein ions in the gas phase is a challenging analytical procedure and the implementation of electron or photon based dissociation techniques such as Electron Capture Dissociation (ECD) and UVPD offers advantages over existing options, including a greater fragmentation efficiency, retention of labile modifications on the protein backbone and the ability to gain insights into the three dimensional folding of the protein by measuring residue-specific fragmentation yields on proteins sprayed from native solutions. Overall, the results gained by ECD top down mass spectrometry are complementary to more routine mass spectrometric measurements already available in Allergan and can provide additional understanding of the structure of the biopharmaceutical of interest.[1][2–4][5]

http://www.mbc.manchester.ac.uk

Entry Requirements:
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Partnership. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

[1] K.L. Fort, C.N. Cramer, V.G. Voinov, Y. V Vasil’ev, N.I. Lopez, J.S. Beckman, A.J.R. Heck, Exploring ECD on a Benchtop Q Exactive Orbitrap Mass Spectrometer., J. Proteome Res. 17 (2018) 926–933. doi:10.1021/acs.jproteome.7b00622.
[2] B. Bellina, J.M. Brown, J. Ujma, P. Murray, K. Giles, M. Morris, I. Compagnon, P.E. Barran, UV photodissociation of trapped ions following ion mobility separation in a Q-ToF mass spectrometer, Analyst. 139 (2014) 6348–6351. doi:10.1039/C4AN01656D.
[3] U.H. Mistarz, B. Bellina, P.F. Jensen, J.M. Brown, P.E. Barran, K.D. Rand, UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides, Anal. Chem. 90 (2018) 1077–1080. doi:10.1021/acs.analchem.7b04683.
[4] Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor
Inês S Camacho, Alina Theisen, Linus O Johannissen, L Aranzazú Díaz-Ramos, John M Christie, Gareth Jenkins, Bruno Bellina, Perdita Barran, Alex R Jones 2019 Proceedings of the National Academy of Sciences 116 4 1116-1125
[5] S.R. Harvey, M. Porrini, A. Konijnenberg, D.J. Clarke, R.C. Tyler, P.R.R. Langridge-Smith, C.E. Macphee, B.F. Volkman, P.E. Barran, Dissecting the dynamic conformations of the metamorphic protein lymphotactin, J. Phys. Chem. B. 118 (2014) 12348–12359. doi:10.1021/jp504997k.

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