Hart, Sarah Ruth; (2003) Analysis of protein phosphorylation sites using affinity enrichment and mass spectrometry. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Reversible modification of proteins by phosphorylation of serine, threonine and tyrosine residues is the most common post-translational modification, which is estimated to occur in 30-90% of the cellular expressed protein component at any one time. Phosphorylation can alter proteins' subcellular distribution, enzymatic activity and specificity. Altered protein phosphorylation may correlate with disease states such as cellular transformation. The analysis of phosphorylated proteins is therefore of vital importance to the field of biology and in particular signal transduction. Protein phosphorylation sites are increasingly investigated using mass spectrometric methods, exploiting the inherent accuracy and sensitivity of these methods. However, the presence of unphosphorylated peptides in enzymatic digests of proteins causes ion suppression of phosphopeptides, reducing the effective sensitivity of detection; this sensitivity is further decreased by the relative lability of the phosphate moiety in the mass spectrometer and the occurrence of sub-stoichiometric modification, which together further reduce the achievable sensitivity. This study has examined techniques for the analysis of protein phosphorylation sites, with particular emphasis upon mass spectrometry. The technique of immobilised metal ion affinity chromatography (IMAC) was investigated in detail as a method suited to phosphorylation site analysis. IMAC exploits the relatively specific affinity of phosphorylated peptides for metal ions, particularly Fe(III), to isolate phosphopeptides upon a solid-phase affinity matrix, separating the suppressing non- phosphorylated component and allowing improved detection of phosphorylated peptides. Conditions for the application of IMAC to phosphopeptide segregation have been established and applied. Using IMAC, protein phosphorylation site identification of both standards and signal transduction mediators has been carried out. Apparent sequence-specific binding of phosphorylated and non-phosphorylated peptides to IMAC resins has been found and investigated. IMAC methodology has been further improved to optimise phosphopeptide analysis using mass spectrometry. The developed methods have clear utility for phosphorylation site analysis, which is vital to the understanding of signal transduction.

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