MALDI-MS investigation of skin and its response to irritants and sensitisers.

There is increasing interest in in-vitro methodology for testing chemical toxicity, one of the drivers for this is European legislation; specifically Directive 76/768 EEC, which prohibits animal testing in the cosmetic industry. In skin toxicological testing, an alternative methodology to in-vivo irritancy experiments already available uses a synthetic skin model and involves measurement of cell viability and release of cytokine interleukin (IL)-1a. There is currently no fully validated in-vitro test for sensitization, although there are a number that are in development. Some of these include the human cell line activation test (h-CLAT) (Sakaguchi et al., 2010), the direct peptide reactivity assay (DPRA) (Gerberick et al., 2009) and the myeloid U937 skin sensitization test (MUSST) (Ade et al., 2006). This study utilises ex-vivo human skin as an initial platform for method development. Areas of analysis have included the lipidomic and proteomic responses of ex-vivo human skin to sensitizers and irritants.Matrix assisted laser desorption ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) was used to identify peptides and lipids in-situ. Ion mobility separation allowed for discrimination between isobaric species due to the selection of specific precursor ions and cleaner MS/MS spectra. Many of the peptides identified belonged to serum albumin, keratin and collagen protein families, which are known to be abundant within human skin. Lipids identified included: sphingomyelin, glycerophospholipids, ceramide species anddi/triglycerides.In skin studies performed in other research groups, sphingomyelin and ceramide species were found to change expression in response to initiation of inflammation, thus making them of biological significance when investigating lipidomic responses to chemical sensitizers and irritants. Tryptic peptide MALDI-MS images have shown differences in human skin, as a result of chemical exposure. MALDI-MS images at 150pm and 30pm resolution have provided information on the localisation of detected species. Changes in expression of species in treated and untreated samples, as well as between different layers of human skin have also been visualised via multivariate statistical analyses. These analyses of ex-vivo human skin have demonstrated that MALDI-IMS-MS is a technique which can be used to detect changes in protein/peptide and lipid profiles in response to sensitisers and irritants. The technique also allows for the localisation of species detected within the different layers of human skin.