Strategies for the examination of Alzheimer's disease amyloid precursor protein isoforms.

The principal aim of this research project has been the utilisation of various proteomic techniques in the investigation of the Alzheimer's disease amyloid precursor protein (APP) isoforms, namely APP[695], APP[751] and APP[770]. One of the most noticeable pathological characteristics of Alzheimer's disease is the presence of neuritic plaques in brain tissue. The chief protein constituent of neuritic plaques is the beta amyloid peptide. This peptide is proteolytically cleaved from APP, as such the interest in APP isoforms is great and a rapid detection method for the presence of each isoform would be a huge advantage to the research effort with regards to the determination and concentration in both diseased and non-diseased states. Two-dimensional gel electrophoresis and peptide mass fingerprinting are two of the most important techniques in the proteomics arena and both are investigated fully in this work. Retinoic acid induced Ntera 2 cells, derived from a human teratocarcinoma cell line, were the in vitro source of APP. Initial isolation of APP was performed by immunoprecipitation, using a monoclonal antibody raised to amino acids 1-17 of the beta-amyloid peptide sequence, which is present in all three alpha secretase cleaved isoforms of interest. The next step was to separate whole APP into its isoform components by two-dimensional gel electrophoresis. The resulting protein spots were then subjected to peptide mass fingerprinting employing the different digest reagents, trypsin, endoproteinase Asp-N and formic acid. Initial distinction between the APP isoforms could be seen upon examination of theoretical in silica digests using the various digest reagents mentioned. The in silica digests revealed peptides unique to each isoform that in theory could be used as indicators of isoform presence

Label-free Quantitative Proteomics and Substrate Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases and nucleases in six human skin explants and a 3D living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C (ADH1C), the most abundant phase I XME in human skin, and glutathione S-transferase pi 1 (GSTP1), the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (CYP) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate CYPs were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin (TXN) and peroxiredoxin-1 (PRDX1). This systematic determination of functional equivalence between human skin and LabSkin is a key step towards the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. Significance Statement The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing

Label-free Quantitative Proteomics and Substrate Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in ex Vivo Human Skin and a Human Living Skin Equivalent Model

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases and nucleases in six human skin explants and a 3D living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C (ADH1C), the most abundant phase I XME in human skin, and glutathione S-transferase pi 1 (GSTP1), the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (CYP) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate CYPs were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin (TXN) and peroxiredoxin-1 (PRDX1). This systematic determination of functional equivalence between human skin and LabSkin is a key step towards the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. Significance Statement The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing