Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.769427
Title: Elucidating protein/small-molecule interactions through direct and indirect mass spectrometry approaches
Author: Chavas, Thomas Edmond Joseph
ISNI:       0000 0004 7657 676X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Abstract:
The elucidation of protein/small-molecule (SM) interactions continues to garner considerable interest from the research community, not least because of its potential downstream applications in the development of new drugs. In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as an extremely powerful tool to study proteomes and, combined with activity-based protein profiling (ABPP), should enable the study of protein/SM interactions in a native environment. However, it appears that the majority of studies in the field fail to report unambiguous MS/MS characterisation of SM/peptide adducts. We argue that this is due to the fact that the current tools for data analysis do not accommodate for the non-canonical behaviour of such adducts during sample processing and MS/MS analysis. In the first part of this work, we develop an indirect strategy based on stable-isotope labelling to study the interactions of G9a with a photoaffinity probe. The inconsistent results obtained were initially thought to be due to poor affinity of the probe for its target protein. Thus, a second photoaffinity probe bearing a diazirine photocrosslinker derived from biotin was designed and synthesised, with the aim of resolving its interaction with avidin. Unfortunately, the study of this model system was not successful; this is most likely due to the poor photocrosslinking yields of the diazirine moiety. The second part of this work was focused on the use of an in-house algorithm to facilitate the MS/MS characterisation of SM/peptide adducts using unconstrained sequencing. This allowed us to identify several sites of interaction between a published sulfonyl fluoride activity-based probe (ABP) and its target proteins. In the process, we uncovered that the probe undergoes chemical displacement and/or hydrolysis from the protein's active site during sample processing. Isotopic labelling strategies allowed us to propose a mechanism for the hydrolysis of the adduct involving neighbouring-group participation. Finally, the ABP's lability was leveraged to specifically label the active site and facilitate its MS identification.
Supervisor: DiMaggio, Peter ; Fuchter, Matthew ; Brown, Bob Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.769427  DOI:
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