Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.755478
Title: Strategies for characterisation of the unexplored human phosphoproteome
Author: Hardman, G. E.
ISNI:       0000 0004 7428 4720
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Abstract:
Post-translational modification of proteins by addition of a phosphate group serves as a means of rapidly regulating protein function, and thus has vital roles in all aspects of cell biology. Phosphorylation of serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the function of such modification in human cells. However, there is growing evidence that phosphohistidine (pHis) may also have a role in diverse cellular processes in mammalian systems. Characterisation of pHis is compromised by the inherent instability of the acid-labile phosphoramidate bond, meaning there is a severe lack of suitable experimental strategies to pinpoint the presence of pHis in proteins. The acidic conditions integral to current phosphoproteomics workflows are incompatible with analysis of acid-labile pHis. In this work, chemical phosphorylation was used to generate a set of model pHis-containing peptides, for which stability of the histidine phosphorylation across a range of pH values and at elevated temperature was assessed. The pHis-containing peptides were further used to evaluate various existing enrichment strategies for their suitability for pHis. None of the enrichment methods tested for this work that had previously been used for enrichment of serine/threonine phosphopeptides enabled recovery of pHis peptides. Subsequently, a strong anion exchange fractionation method has been developed, which permits separation of pHis-containing peptides from non-phosphorylated equivalents with salt gradient elution at non-acidic pH. Fractions at the end of the gradient are significantly enriched for phosphopeptides. Crucially, the unbiased phosphopeptide enrichment by strong anion exchange (UPAX) method enabled recovery of pHis-peptides from complex cell lysates. The UPAX method with characterisation by LC-MS/MS has revealed extensive phosphorylation of histidine, lysine, arginine, aspartate and glutamate in human cell extract, including 310 pHis sites and over 1000 sites of lysine phosphorylation. Remarkably, the extent of non-canonical phosphorylation far exceeds that of phosphotyrosine, exposing the previously underappreciated diversity of the human phosphoproteome. The results of this work highlight possible roles for pHis and other sites of non-canonical phosphorylation in human cells. Identification of pHis at a conserved residue within the catalytic domain of eukaryotic protein kinases, suggests histidine phosphorylation may act as a general mechanism for kinase regulation. Application of the UPAX strategy for unbiased phosphoproteomics analysis has opened up diverse avenues for exploring roles and regulation of non-canonical phosphorylation sites in any proteome.
Supervisor: Eyers, Claire Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.755478  DOI:
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