Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733121
Title: The development of a cellular model and mass spectrometry workflow for characterising protein tau in Alzheimer's disease
Author: Da, Zi Ran
ISNI:       0000 0004 6496 1126
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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Abstract:
Background: Neurofibrillary tangles (NFTs) is one of the two ‘hallmarks’ of Alzheimer’s disease (AD). The main constitute of these tangles are known as the paired helical filaments (PHFs), which are essentially abnormal aggregate of microtubule associated protein Tau. It has been known for many years that the hyper-phosphorylation of protein Tau is a persistent feature in NFTs. However, whether hyper-phosphorylation of Tau is a cause or an effect to aggregation remains elusive. Mass spectrometry can now serve as an alternative to immunoblotting analysis for post translational modifications (PTM) study. Results: We developed a mass spectrometry method targeting on Tau protein for phosphorylation study. For a decent MS characterisation run, HEK 293 cells confluent in 5-10 plates (50 cm2) are required to be fed to the sample processing procedure. Typically, it takes 4-6 days to prepare the cells into MS samples. The targeting approach was able to discover peptides less intense by two orders of magnitude than that spotted using the general discovery method. The application of the targeted method on the soluble Tau recovered from HEK 293 cells allowed the solid discovery of three additional Tau phosphopeptide (only one phosphopeptide was discovered previously). Furthermore, for the development of the MS targeted method and high purity MS sample preparation, an efficient recombinant Tau protein production (in E.coli) protocol and a purification workflow for phosphorylated and untagged soluble/functional Tau protein (from HEK 293 cell line) was developed respectively. Conclusion: The Tau protein targeted MS method allows solid identification of additional phosphopeptides at two orders of magnitude lower peak intensity. The compatible purification protocol for soluble Tau could be applied to mammalian cells and tissue that expresses wild type and untagged Tau protein.
Supervisor: Heng, Jerry ; DiMaggio, Peter Sponsor: Chemical Engineering
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.733121  DOI:
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