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Method for identification of low soluble, biopersistent susts (GBS) - comparison of intratracheal installation and subacute inhalation

: Creutzenberg, Otto H.; Hansen, Tanja; Schuchardt, Sven; Tillmann, Thomas; Knebel, Jan

Naunyn-Schmiedebergs archives of pharmacology 389 (2016), Supplement 1, pp.S47, Abstract 189
ISSN: 0028-1298
ISSN: 1432-1912
Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (Annual Meeting) <82, 2016, Berlin>
Network Clinical Pharmacology Germany (Annual Meeting) <18, 2016, Berlin>
Fraunhofer ITEM ()

Cytochrome P450 enzymes and transporters are important for the turnover of pharmaceutical compounds. Their expression levels and activity influence bioavailability and convey drug-drug interactions. Moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. Overexpression of export transporters in tumors can lead to multiple drug resistance. However, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. Antibody – based analysis of cytochrome P450 enzymes and transporters is challenging due to their sequence homology. Therefore, we developed a test system for protein quantification which combines the sensitivity of immune precipitation and the specificity of mass spectrometry: This method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. One peptide from each protein, which can be assigned unambiguously, is identified via tandem MS and quantified by means of an isotope labeled reference. Prior to MS-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. These epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. The major advantage of this method is that whole cell or tissue lysates – without preparation of microsomal fractions – can be used for quantification by LC-MS. Also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. Currently, we have established assays with 25 antibodies covering 15 Cytochrome P450enzymes and 17 transporters in eight different species – in total 92 proteins. References: Gottesman MM, Fojo T, and Bates SE. 2002. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature reviews. Cancer Eisen D, Planatscher H, Hardie DB, Kraushaar U, Pynn CJ, Stoll D, Borchers C, JoosTO, and Poetz O. 2013. G protein-coupled receptor quantification using peptide group-specific enrichment combined with internal peptide standard reporter calibration. Journal of proteomics: Hoeppe S, Schreiber TD, Planatscher H, Zell A, Templin MF, Stoll D, Joos TO, and Poetz O. 2011. Targeting peptide termini, a novel immune affinity approach to reduce complexity in mass spectrometric protein identification. Molecular & cellular proteomics Weiss F, Schnabel A, Planatscher H, van den Berg BH, Serschnitzki B, Nuessler AK, et al. 2015. Indirect protein quantification of drug-transforming enzymes using peptide group-specific immune affinity enrichment and mass spectrometry. Scientific reports Weiss F, van den Berg BH, Planatscher H, Pynn CJ, Joos TO, Poetz O. 2014. Catch and measure-mass spectrometry-based Biochimica et biophysica acta.