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.

Publications

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