Analysis of the dynamic sulfotyrosine proteome.

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The survival of an organism depends upon the ability of different cell types to communicate with each other by assembling the correct complexes of proteins at the correct time in the correct place. One way this is achieved is to use the tricks of chemistry and biological catalysts (enzymes) called tyrosyl protein sulfotransferases, or TPSTs, to change the biological properties of polymers, such as proteins. TPSTs drive this process by adding small charged chemicals as a means of rapid (usually) reversible regulation. These events, more accurately called 'post-translational modifications', or PTMs, act as switches to change information flow and dictate the types of different biological outcomes elicited, such as cell movement, growth, survival or death. Our proposal aims to exploit new tools and proteomics technology (using mass spectrometry, which looks at all proteins in cells in an unbiased way) to evaluate the addition of a specific chemical group, called sulfate, to proteins. Currently, the analysis of sulfation is unfocused, it attracts little strategic funding, and it is difficult to manipulate or study in a holistic manner, making efforts to study its global significance challenging. Since it underpins so much basic biology, yet detailed information as to the mechanism of this regulatory mechanism is still lacking, sulfation research requires concerted strategies to develop ways of analysing, compiling and distributing, the large amounts of biological data pertaining to how protein sulphation regulates cellular and acellular biology. Indeed, technology-based approaches for the analysis of a different chemical group on proteins, phosphate, led to a revolution in our understanding of how cells communicate, and has been critically important for biologists working in the areas of structural biology, cell signalling and communication and drug design for over 40 years, with knock-on effects on biotechnology, pharmaceutical industries and clinical intervention across the world. To rapidly advance our understanding of cellular protein tyrosine sulfation (sTyr), our aims will be achieved through related, but distinct, work packages. These are:WP1: Biochemical analysis of sTyr (and sThr/sSer) site-specificity in a variety of peptides and proteins WP2: sTyr isolation and mass spectrometry-based quantitative sTyr analysis from complex cell-derived mixturesWP3: Optimise cell-based approaches to manipulate TPST1/2 and perturb sTyr content in mammalian cellsWP4: Computational analysis of mass spectrometry data and dissection of tyrosine sulfation networks for public distribution and biological inferenceWP5: A workflow for analysis of new cellular roles associated with Tyr sulphation