Analysis of post-translationally modified proteins using mass spectroscopy

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It is simply not true to say that the function of a gene is determined by the sequence of the amino acids for which it codes. The nascent polypeptide may be subjected to a wide array of post-translational modifications. In many cases, these modifications either are essential for the correct functioning of the protein, or are an integral part of the mechanism by which the activity of the protein may be modulated. The identification of the sites of PTM and understanding the dynamics of the modification process are therefore essential prerequisites to understanding the control of protein activity. We apply for funds to purchase an Applied Biosystems 4000 Q-TRAP Pro mass spectrometer. This instrument will be used to analyse proteins and peptides from complex mixtures for the purpose of mapping post-translational modifications (PTM) in proteins with high sensitivity in medium throughput. Funding is only requested for the purchase of the spectrometer itself. Ancillary equipment required for its daily operation (e.g. a capillary liquid chromatography system, nitrogen gas generation and workstation computing for data analysis) already exist with the Biomolecular Analysis Core Facility within the Faculty of Life Sciences. The Core Facility in which the instrument will be housed does, however, currently lack the ability to undertake analysis of PTMs in proteins available at low concentration. The proposed instrument will both extend and enhance our mass spectrometry capabilities. The machine will be used extensively throughout the faculty and, in addition, by users from other Universities and from industry. The projects that will exploit this system mainly involve research in basic biomedical sciences using mammalian cells or model organisms such as yeast. The instrumentation to be purchased allows these projects to take advantage of proteomics approaches. Specifically, and as outlined within this application, the machine will be used to analyse PTMs in the following areas: phosphorylation and methylation of transcription factors that play vital roles in the regulation of activity; modification (phosphorylation and hypusinlation) of translation factors; regulation of Golgi and dynein function through specific modification; characterisation of the redox state of ER proteins; systems approaches to understanding metabolic flux and the development of bioinformatics support.