Claire Eyers

Claire Eyers

Talk Title: Unbiased enrichment of the phosphoproteome gives unprecedented insight into novel human signalling mechanisms through pHis, pAsp, pLys, pArg


Claire E. Eyers is Professor of Biological Mass Spectrometry in the Institute of Integrative Biology (IIB) at the University of Liverpool (UoL) and co-Director of the Centre for Proteome Research (CPR).

Having obtained a PhD (2002) in Biochemistry from the University of Dundee (Prof. Sir P. Cohen), she undertook postdoctoral studies at the University of Colorado, Boulder (Prof. N. Ahn) and then in the Michael Barber Centre for Mass Spectrometry, University of Manchester (Prof. S. Gaskell), where she became Acting Director (2009–2013). Her research exploits biophysical and biochemical methodologies to understand the structure and relevance of post-translational modifications (PTMs) and she has established expertise in the development of mass spectrometric (MS)-based methods, and more recently ion mobility spectrometry (IMS), for the investigation of proteins and glycans.
CEE has held an independent American Heart Association (AHA) fellowship, for which she was awarded an AHA postdoctoral prize, and a Royal Society Dorothy Hodgkin Fellowship (2007-11). She is currently Treasurer of the British Mass Spectrometry Society, co-organises the BMSS Ion Mobility Special Interest group, and is an active STEM ambassador.


Keywords: mass spectrometry, phosphoproteomics, histidine, strong anion exchange, phosphorylation

Authors: Gemma Hardman1, Simon Perkins2, Sian Canadine2, Philip Brownridge1, Andrew R. Jones2, Claire E. Eyers1.

1 Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, United Kingdom
2 Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, United Kingdom

With growing evidence for phosphohistidine in mammalian cell signalling, there is a pressing need for development of analytical strategies that permits high-throughput characterisation of these acid-labile phosphorylation sites from complex biological systems. Standard phosphoproteomics workflows that rely on acidic conditions for phosphopeptide enrichment are not suitable for characterisation of these types of phosphopeptides. We have develop an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX) that permits enrichment of these acid-labile phosphopeptides for characterisation by LC-MS/MS. Not only have we identified ~300 novel sites of endogenous His phosphorylation in human cell extracts, we have also demonstrated even more extensive phosphorylation of Asp, Arg and Lys in human cells at levels that exceed phosphotyrosine. This study represents the first unbiased phosphoproteomics investigation in any biological system. Critically these data reveal a paradigm shift in the way that we currently understanding mammalian signalling, opening up diverse avenues for investigation of the roles and regulation of acid-labile phosphorylation sites.

Tryptic peptides of α-/β-casein and histidine phosphorylated myoglobin, generated by chemical phosphorylation of myoglobin with potassium phosphoramidate, were used for assessment and optimisation of phosphopeptide enrichment strategies. For enrichment by SAX, peptides were bound to a PolySAX LP column under non-acidic conditions, and bound peptides eluted with a gradient of triethylammonium phosphate. Samples were analysed using a Waters nanoAcquity LC coupled to a Bruker AmaZon mass spectrometer, operated in top-3 CID/ETD mode, or an Orbitrap Fusion Tribrid instrument using HCD and neutral loss triggered EThcD. MASCOT/ptmRS through Proteome Discoverer was exploited for data anlaysis.

Currently employed phosphopeptide enrichment strategies (TiO2, calcium phosphate precipitation, hydroxyapatite) were all unsuccessful for enrichment of phosphohistidine-containing peptides. Optimisation of SAX for phosphopeptide separation permitted identification of ~300 novel sites of histidine phosphorylation. Moreover, we characterised even more extensive phosphorylation of phosphoarginine and phosphoaspartate and ~600 novel sites of phospholysine, exceeding the total number of sites phosphorylated on tyrosine identified in these studies.

The UPAX method overcomes challenges associated with enrichment of phosphopeptides containing acid-labile phosphoamino acids, and can additionally be used for the more traditionally studied pSer/pThr, pTyr. This unbiased phosphoproteome analysis has revealed extensive phosphorylation of His, Asp, Arg and Lys in mammalian cells to levels that likely match or exceed pTyr, and will undoubtedly revolutionise our understanding of mammalian cell signalling mechanisms.