The Mass Spectrometry facility
Biological Mass Spectrometry (Bio-MS) has benefited through the acquisition of a new Orbitrap Elite mass spectrometer. This system is highly sensitive and specific, providing new levels of mass accuracy and signal resolution that has revolutionised our capabilities to look at protein modifications and to compare complex mixtures of proteins. The facility is one of the largest of its type in Europe, and it has a complete suite of mass spectrometers and sample preparation technologies that supports work within the Centre. Other mass spectrometry equipment includes; Thermo Velos Pro; Applied Biosystems 4000 Q-Trap; Bruker Ultraflex II MALDI TOF-TOF; Bruker HCT Ultra; Agilent 7890GC with 5975C mass spectrometric detector; Agilent 6520 Q-TOF; Waters LCT. Sample preparation technologies include; 1 and 2D gel electrophoresis, preparative PAGE (Expedeon GelFree 8100), isoelectric focusing (Agilent Offgel); standard and micro scale HPLC purification systems). The facility‟s primary role is the identification, characterisation, and quantification of proteins, and it also supports the profiling and quantification of metabolites. Its main focus is the quality of the results produced and, as well as providing established methodologies, it is also involved in developing new methods in areas such as high resolution separations, characterising modifications, and absolute protein quantification. The facility is more than a collection of instruments and its aim is to provide the highest level of support for the researchers who use the facility. To achieve this, it supports all aspects of the research process from project specific advice on experimental design and sample preparation, to performing the analyses and aid in data interpretation. An internal focus of the facility is now to push the limits of these technologies within the clear remit of good analytical science, ensuring that the highest quality, robust and trustworthy data are provided. The facility has also benefitted from a hybrid experimental officer/post-doc to facilitate the use of the facility by groups expanding their research in to new biochemical areas.
Supporting the Cell-Matrix Centre
Gilmore: The facility has helped to develop methods to link mass spectrometry to light microscopy via the use of fluorescent tags. Moreover proteomics analysis was instrumental in identifying new targets of p38 signalling in the adhesion-control of apoptosis in breast epithelia.
Humphries: Improving on our existing work elucidating the composition of adhesion complexes, the facility is now also helping in developing quantitative methods of looking at the dynamics of the complex assembly and disassembly as well as looking at the phosphorylated signalling pathways triggered on adhesion.
Lennon/Streuli: The facility has supported work looking at defining the ECM for a number of years and is now in the process of expanding these methods out into more clinical environments. It is currently working with Lennon on the identity of the glomerular matrisome in kidney, with the aim of discovering how the ECM changes in inherited kidney diseases that occur in children. In addition, together with Streuli, it is employing similar techniques for proteomic analysis of the matrisome in breast epithelia, with the aim of identifying specific ECM proteins that characterise mammographically dense breast tissue – this work may lead to novel mechanisms of susceptibility to breast cancer.
Thornton: Developing new robust methods of absolute quantification of mucin domains. These approaches cannot only be used to investigate mucin tailoring during disease, but can also for the basis of higher quality quantification and clarification of mucus in the clinical environment. The facility is also supporting efforts to link together next generation sequencing approaches, such as RNA Seq, and mass spectrometry to investigate and characterise the biology of pathogens.