ProteinsAlthough much attention is focussed on genes, proteins are the molecules that perform most of the functions of an organism, and so the study of protein biochemistry is essential for understanding the fundamental processes of life. It is only in the last 20 years that mass spectrometry of proteins has become practical and it is now an essential tool in protein analysis. There are many uses of mass spectrometry in protein biochemistry, but perhaps the most common is in the identification of unknown proteins. The methodology has been refined from the starting point where individual purified proteins could be identified, to the current situation where several thousand proteins can not only be identified, but also quantified, in a single analysis. We use these techniques in three main areas of research:
Characterisation of Protein ComplexesThe function of most proteins is expressed, not as individual molecules, but as part of multiprotein complexes. A critical first step to understanding these biochemical processes is to identify the proteins that make up the functional complex. We use this approach extensively to study protein complexes involved in many different processes, for example in the two references below; the regulation of RNA degradation; and gene silencing through the modification of chromatin structure.
Frenk, S., et al. (2014). The Nuclear Exosome Is Active and Important during Budding Yeast Meiosis. PLoS One, 9(9), e107648. doi: 10.1371/journal.pone.0107648
Tavares, L., et al. (2012). RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.
Cell, 148(4), 664-678. doi: 10.1016/j.cell.2011.12.029
Post-translational modifications (PTMs)Most proteins have their primary structures modified in some way after they are synthesised. There are several hundred known modifications, including phosphorylation, glycosylation, ubiquitination, lipidation, poly-ADP-ribosylation. PTMs can dramatically affect protein activity, interactions, localisation and turnover. Since modification of an amino acid usually results in a change of molecular weight, PTMs can potentially be identified, and their sites of attachment determined, by mass spectrometry. This is rarely straightforward, but it is one of the major interests in the Facility. Phosphorylation is of particular interest in signalling as it is a perhaps the most important mechanism of signal transduction within the cell.
Gilley, R., et al. (2012). CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIMEL.
Cell Signal, 24(1), 170-180. doi: 10.1016/j.cellsig.2011.08.018
Zhao, R., et al. (2007). DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH.
PLoS Biol, 5(1), e1. doi: 10.1371/journal.pbio.0050001
Targeted AnalysisMass spectrometry can be used for the quantitation of proteins and PTMs as well as for identification and structural characterisation. Although absolute quantitation is possible, it is more usual to measure changes in the abundance of particular proteins or PTMs under different conditions e.g. increase in phosphorylation of a target site when a kinase is activated. The very high selectivity, sensitivity, dynamic range, and scan speeds of modern mass spectrometers, means that particular proteins or PTMs of interest can be targeted for analysis, even in an extremely complex background, such as a total cell lysate. This allows us to monitor the levels of anything up to several hundred proteins or PTMs in a single analysis, and to see how they change, e.g. following activation of a signalling pathway, in response to a drug treatment, or during the course of cell differentiation.
Ashford, A. L., et al. (2014). A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3beta to phosphorylate cyclin D1 at Thr(286), not Thr(288).
Biochem J, 457(1), 43-56. doi: 10.1042/bj20130461
Brien, P., et al. (2013). p38alpha MAPK regulates adult muscle stem cell fate by restricting progenitor proliferation during postnatal growth and repair.
Stem Cells, 31(8), 1597-1610. doi: 10.1002/stem.1399