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Labelled IMS TAG Proteins for Quantitative Mass Spectrometry Imaging

Almost a quarter of a million pounds has been awarded to a team of scientists at Sheffield Hallam University to develop a new technique that will reduce the need for animal testing in pre-clinical research projects.

The team has been given £244,045 to develop its Mass Spectrometry Imaging (MSI) technology to provide quantitative analysis of the level of protein found in biological tissue.

In previous projects that looked at the level of protein change in tumours following the administration of anti-cancer drugs, Sheffield Hallam researchers found that there was a need to be able to measure the change in protein levels to allow clinicians to have an accurate picture of how tumours respond to treatment. While conventional analysis can only quantify a small number of proteins, MSI is a powerful tool that is used to map different molecules within tissue sections and can produce multiple images of protein responses to medical treatments.

Leading the team, Professor Malcolm Clench of the University's Biomolecular Sciences Research Centre, said, 'If we were to study the activity of 50 proteins within tissue, under the usual method, you would need around 25 mice for testing but by using mass spectrometry imaging, only one mouse would be needed.

'We've had some exceptional feedback from the reviewers and this new funding will help us to improve the accuracy of the data it will produce and we can then look to introduce it into pre-clinical testing on a global scale.'

Sheffield Hallam is one of only five institutions in the country to have been awarded the funding by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) in collaboration with the Engineering and Physical Sciences Research Council (EPSRC).

Dr Vicky Robinson, chief executive of the NC3Rs, said, 'The potential for technological development to replace, reduce and refine the use of animals in science is now well recognised across the research community. Pre-clinical imaging offers an opportunity for researchers to greatly reduce and refine animal use through longitudinal studies and identifying earlier endpoints to reduce suffering.

'However its application is often restricted by limitations with the current technologies available. This strategic funding allows the NC3Rs to target key areas identified by the research community where the development and application of new imaging techniques could have a profound impact on animal use and science.'

About the project

This project aims to develop methodology for the quantitative Mass Spectrometry Imaging (MSI) of proteins in biological tissue. Two non-animal models will be used both of which comprise multi-cellular, three-dimensional (3D) tissue constructs fabricated from human cell lines; a novel type of tumour spheroid model developed in our laboratory and a commercial 3D human skin construct.

Successful development of the methodology will present an alternative to both in vivo and ex vivo testing using animal models to represent human tumours and skin. The developed methodology will also be applicable to animal tissue where the ability to image and quantify multiple non-labelled proteins has the potential to reduce the number of animals used in many procedures, in comparison to conventional techniques employing immunohistochemistry, and immunoflourescence.

MALDI-MS imaging is a novel, label-free imaging technique that can be used to image the changes in multiple protein responses following treatment. We have previously applied MALDI-MS imaging to the study of ex-vivo human skin, 3D cellular skin models as well as human tumours, xenografts and allogenic animal tumour models. One of the major challenges facing MALDI-MS imaging is the quantification of changes in protein response observed in the images. Whilst progress has been made in quantitative analysis of small molecules by MALDI-MS imaging, quantitative analysis of protein images has not been attempted.

We have recently introduced the concept of 'IMS-TAG' proteins for validation of protein identity. IMS-TAG proteins are recombinant proteins produced to contain signature peptides from a variety of proteins of interest that are present in the tissue under study. These peptides are released by enzymatic digestion of the recombinant protein and are used as positive controls for matching accurate mass, ms/ms spectra and ion mobility drift times in imaging experiments.

Here it is proposed to further develop the IMS-TAG idea by producing two 15N labelled IMS-TAG proteins: one containing signature peptides for clinically relevant skin proteins involved in psoriasis and eczema and one containing peptides for those proteins involved in cancer progressions. The enzymatically generated labelled peptides will then be used as internal standards for quantitative MALDI-MS imaging experiments by incorporation of the unlabelled protein into tissue homogenates to create calibration standard arrays and by subsequently spraying cut sections of the homogenate arrays with labelled protein as an internal standard. 

Although this study is being carried out on skin and tumour models the methodology would be directly applicable to all other tissues. Software for the extraction of quantitative information will be developed in collaboration with Waters Corp.

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