The aim of this project is to reduce animal use by improving the utility of tissue engineered constructs through the development of quantitative mass spectrometry imaging (MSI) of proteins in these biomimetic 3D tissues.
Matrix-assisted laser deabsorption/deionisation mass spectrometric (MALDI-MS) imaging is a novel label-free imaging technique that can be used to image the responses of multiple proteins following drug treatment. The technique has been applied in the study of ex vivo human skin, 3D cellular skin models, human tumours, and allogenic animal tumour models. One of the major challenges facing MALDI-MS imaging is that changes in protein responses observed in the images generated cannot be quantified. This project will build on the concept of ‘IMS-TAG’ proteins to overcome this challenge and to validate the identity, and quantity, of specific proteins within biomimetic tissue constructs. These tools will be developed and tested in 3D skin and tumour spheroid models to demonstrate their utility in addressing specific research questions without animals.
Research details and methods
This project will demonstrate the use of MALDI-MS imaging for the quantification of proteins in response to treatments for common skin diseases and cytotoxic anti-tumour agents. It will develop novel ‘IMS-TAG’ proteins labelled with 15N and which contain signature peptides for clinically-relevant skin proteins involved in psoriasis and eczema, and one for proteins involved in responses to anti-tumour agents. These peptides will be released by enzymatic digestion of the ‘IMS-TAG’ protein and will be used as internal standards for quantitative MALDI-MS imaging experiments. Distribution of the target proteins as imaged using MALDI-MS imaging will be validated using traditional immunohistochemistry of the skin and tumour constructs.
Flint LE et al. (2020). Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging. Analytical Chemistry, in press. doi: 10.1021/acs.analchem.0c02389
Palubeckaitė I et al. (2019). Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model. Journal of Mass Spectrometry 55(4):e4461 doi: 10.1002/jms.4461
Lewis EEL et al. (2018). Examination of the skin barrier repair/wound healing process using a living skin equivalent model and matrix-assisted laser desorption-ionization-mass spectrometry imaging. International Journal of Cosmetic Science 40(2):148-56. doi: 10.1111/ics.12446