Labelled IMS TAG Proteins for Quantitative Mass Spectrometry Imaging
Lead Research Organisation:
Sheffield Hallam University
Department Name: Faculty of Health and Wellbeing
Abstract
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 immunoflouresence.
Technical Summary
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. To date, 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 containig 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.
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 containig 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.
Planned Impact
Who will benefit from this research?
The research will benefit all researchers who use animal tissue to study protein expression in response to stress, disease, treatment or exposure to toxicants. The research will be particularly applicable where the distribution of such proteins is usually studied by immunohistochemistry as quantitative mass spectrometry imaging enables the study of multiple proteins in one tissue section. This has huge potential for the reduction of animal usage in scientific research. The choice of non-animal tissue models will demonstrate immediate applicability in the area of skin and cancer research. With respect to tumour models, tumour spheroids are becoming increasingly used in initial drug discovery to responses more closely mimicking in vivo situation than monolayer culture. The ability to simultaneously image drug responses and drug distribution in complex 3D cell systems will reduce the need for animal testing particularly in initial studies, benefiting Pharm industries and academic researchers. The clear commercial and industrial impact of the research to UK based companies is demonstrated by the agreement of Stefiel/GSK (end user) and Waters (Instrument Manufacturer) to collaborate in the project. The project team will disseminate their findings and methodology to a range of audiences from academics, industrial stake holders and the lay public demonstrating the wider applicability of the approach.
How will they benefit from this research?
As well as ethical considerations animals are expensive. Reduction and Replacement of animals will reduce costs and increase competitiveness for companies that adopt these procedures. Mass spectrometry imaging has been widely adopted by the pharmaceutical sector such that developed methodologies could be immediately adopted to existing technology. The pharmaceutical sector, and academic research sector will benefit from this research by a more streamlined method for initial testing of drug distribution vs activity in complex 3D cell systems, reducing animal usage in early stages of drug discovery. At SHU we have been proactive in providing training in MS imaging to companies and research organizations and we will offer via out commercial arm training in quantitative imaging. We are proud to have been able to train 23 PhD students in mass spectrometry, these students have go on to utilize their expertise in laboratories around the world. The two PDRAs employed on this award will become experts in the new technique and hence highly employable.
The research will benefit all researchers who use animal tissue to study protein expression in response to stress, disease, treatment or exposure to toxicants. The research will be particularly applicable where the distribution of such proteins is usually studied by immunohistochemistry as quantitative mass spectrometry imaging enables the study of multiple proteins in one tissue section. This has huge potential for the reduction of animal usage in scientific research. The choice of non-animal tissue models will demonstrate immediate applicability in the area of skin and cancer research. With respect to tumour models, tumour spheroids are becoming increasingly used in initial drug discovery to responses more closely mimicking in vivo situation than monolayer culture. The ability to simultaneously image drug responses and drug distribution in complex 3D cell systems will reduce the need for animal testing particularly in initial studies, benefiting Pharm industries and academic researchers. The clear commercial and industrial impact of the research to UK based companies is demonstrated by the agreement of Stefiel/GSK (end user) and Waters (Instrument Manufacturer) to collaborate in the project. The project team will disseminate their findings and methodology to a range of audiences from academics, industrial stake holders and the lay public demonstrating the wider applicability of the approach.
How will they benefit from this research?
As well as ethical considerations animals are expensive. Reduction and Replacement of animals will reduce costs and increase competitiveness for companies that adopt these procedures. Mass spectrometry imaging has been widely adopted by the pharmaceutical sector such that developed methodologies could be immediately adopted to existing technology. The pharmaceutical sector, and academic research sector will benefit from this research by a more streamlined method for initial testing of drug distribution vs activity in complex 3D cell systems, reducing animal usage in early stages of drug discovery. At SHU we have been proactive in providing training in MS imaging to companies and research organizations and we will offer via out commercial arm training in quantitative imaging. We are proud to have been able to train 23 PhD students in mass spectrometry, these students have go on to utilize their expertise in laboratories around the world. The two PDRAs employed on this award will become experts in the new technique and hence highly employable.
