Development of sensitive proteomic approaches for protein tyrosine phosphorylation detection.

Abstract

The elucidation of the complex array of cell signalling cascades is imperative for a deeper understanding of cell biology in both physiological and patho-physiological states. Extensive biochemical characterisation of signalling networks has revealed the importance of post-translational modifications (PTMs), particularly phosphorylation. Signalling via protein phosphorylation occurs across homeostatic proliferative, differentiative and anti-apoptotic events. Dysregulation of the kinase signalling pathways as well as mutations in kinases involved in phosphorylation have been implicated in a number of pathologies such as cancer or immune deficiencies. While it is estimated that 50% of all proteins are phosphorylated during their lifetime, phosphorylated proteins are present in relatively low abundance compared to their non-phosphorylated counterparts. The rarity of phosphorylation, which occurs on serine, threonine and tyrosine residues, has prompted the development of sensitive approaches to improve phosphorylation characterisation. Proteomic-based strategies offer novel approaches to overcome the limitations of currently available strategies for phosphoprotein analysis. The research presented within describes the development of proteomic-based methodologies for phosphotyrosine identification, quantitation and characterisation. These methods utilise the antiphosphotyrosine. Antibody 4G10 along with other MS-compatible approaches for phosphotyrosine enrichment prior to MS analysis. Methods for more targeted phosphoprotein analyses involved coupling of 4G10 covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. These 4G10-coupled beads successfully enriched tyrosine phosphopeptides derived from tryptic phosphoprotein digests for identification and characterisation of phosphopeptides using MALDI-TOF/TOF MS analysis. The limited capacity of the magnetic bead approach for analysis of more complex samples necessetated the development of a more global proteomic strategy for tyrosine phosphorylation analysis. A global strategy that provides not only qualitative pTyr information but also shows quantitative changes that occur with pTyr signalling is imperative for detailed signalling cascade analyses. The global approach presented here utilised the 4G10 Ab/bead approach as well as Hydrophilic interaction chromatography (HILIC) for the enrichment of pTyr peptides from complex samples isotopically-labelled to quantify tyrosine phosphorylation after LC-MALDI-TOF/TOF MS analysis. Aspects of this approach were modified to improve phosphopeptide detection and characterisation, including the development of a novel optimised matrix-deposition strategy for LCMALDI-TOF/TOF MS. The strategy, termed EZYprep LC, allowed the effective use of the atypical 2,5-DHB matrix with phosphoric acid to improve phosphopeptide ionisation and subsequently identify and characterise more phosphorylation sites on phosphoprotein samples compared with LC-ESI-IT-MS/MS. Another aspect of the global strategy was the development of a modified isotope protein coded label strategy (modified ICPL). The optimised ICPL approach ensured quantitative information from a larger sub-set of peptides after tryptic digest of complex samples. The improved ability to quantify using this approach was highlighted by a comparative analysis of complex cell lysates labelled using the conventional ICPL strategy and the modified ICPL strategy. The modified ICPL labelling strategy identified more proteins and provided more quantitative information that the conventional ICPL methodology. As such, the global phospho-tyrosine strategy, combined the modified ICPL labelling and 4G10 Ab/bead enrichment with peptide fractionation and MALDI-TOF/TOF MS analysis, was subsequently utilised to identify and quantify tyrosine phosphorylation occurring in insulin-stimulated insulin receptor A- and B-subtypes.