About the Project
Background – Native MS provides an approach for intact protein analysis1 with relevance to drug discovery2 and increasingly used to analyse mAbs as well as other biologics.3,4,5 With appropriate sample preparation, use of aqueous volatile buffers, and suitable tuning of the instrument, it is possible to transfer weakly associated complexes from solution into the gas-phase of a mass spectrometer and obtain insights into complex stoichiometry and protein structure.6 To date there have been only a handful of reports in the literature employing native MS for intact mass and Drug to Antibody Ratio (DAR) analysis of cysteine-linked ADCs.7,8
We have recently developed a method to monitor DARs with native direct infusion MS by addition of the charge reducing agent triethylammonium acetate (TEAA) which preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination.9 We have also started to examine the use of native mass spectrometry from crude cell lysates, thus minimizing sample preparation steps.
Aims – In this project we shall explore the use of direct infusion native IM-MS coupled with topdown sequencing using samples from crude cell lysates of intact ADCs to develop a high throughput screening platform for ADCs and report the stoichiometry of conjugation, as well as the effect of conjugation on native fold. This method will be further applied to determine the structure of enzymes, and to map the altered residues and the effect of these on the active fold.
Year 1 – Training on instrumentation and then improve existing protocols for native MS of mAbs and ADCs, and apply to crude cell lysates
Year 2 – Develop top down sequencing via ECD and Photoionsiation as well as CID for ADCs
Year 3 – Apply native MS and top down to crude cell lysates to enzyme preps and develop data warehouse for top down data
Year 4 – Apply methods to enzyme cascades and to other bioconjugates.
We welcome applications from graduates with a good UK honours undergraduate degree (1st class or a high 2i), or a first degree with an additional masters degree or international equivalent, in chemistry, biochemistry, biology or another aligned science subject. Applicants should be looking for a challenging, interdisciplinary research training environment.
All applicants should send their CV and covering letter to Dr Ian Rowles (CBNM Project Manager) [Email Address Removed]. Applications will be reviewed as they are received until a candidate is selected; therefore candidates are encouraged to apply early.
Funding Notes
This is a 4 year studentship jointly funded by the EPSRC, AstraZenca and the University of Manchester, covering all fees and stipend (£14.777 in September 2018). Open to UK/EU applicants only.
The start date for this PhD prgramme will be September 2019.
References
1. R. Beveridge et al., Nat. Commun. 2016, 7, 12163
2. K. J. Pacholarz et al., Chem. Soc. Rev. 2012, 41, 4335
3. K. J. Pacholarz et al., Angew. Chem. Int. Ed. 2014, 53, 7765
4. R. Upton et al., The Application of Mass Spectrometry for the Characterization of Monoclonal Antibody-Based Therapeutics, 2016
5. R. Upton et al., Anal. Chem. 2016, 88, 10259
6. J. D. Eschweiler et al., J. Am. Soc. Mass Spectrom, 2017, 28, 1991
7. J. F. Valliere-Douglass et al., Anal. Chem. 2012, 84
8. J. Chen et al., Anal. Chem. 2013, 85, 1699
9. K. J. Pacholarz, P. E. Barran, EuPA Open Proteom., 2016, 11, 23
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