The primary role of the Facility is to apply state of the art techniques in mass spectrometry to address important biological questions, within the remit of the Institute
Peptide mapping by LC-MS/MS
Accurate mass measurement of an intact protein (<10ppm) or small molecule (<1ppm)
Quantification of modified bases (e.g. 5-methylcytosine and 5-hydroxymethylcytosine) in DNA
Protein identification and relative quantification
Identification and localisation of protein posttranslational modifications
Development of quantitative LC-MS/MS assays for the analysis of specific proteins/small molecules in complex matrices
Expertise & Advice
The staff of the facility have many years experience in chemical, biochemical and physical analysis of many different types of biomolecules, including proteins, nucleic acids, carbohydrates, and small molecules. We use mass spectrometers, to detect, quantify and obtain structural information on biomolecules, which are often present in highly complex samples and in extremely small amounts.
Much of our work is focused on proteins, the molecules that perform most of the functions of an organism. There are many uses of mass spectrometry in protein biochemistry, but perhaps the most common is in the identification of unknown proteins. It is now possible to identify and quantify several thousand proteins in a single analysis. We use these techniques in three main areas of research:
Characterisation of Protein Complexes The 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 e.g. Tavares et al. (2012).
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. Phosphorylation is of particular interest in signalling as it is the most important mechanism of signal transduction within the cell. Odle et al. (2019), Sale et al. (2019).
Targeted Analysis Mass spectrometry can be used for the quantitation of known 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, or in response to a drug treatment, etc. Tsolakos N, et al. (2018).