Sensitive and Specific Proteomic Identification and Quantitation of Murine Cytochrome P450 Enzymes and Histone Post-Translational Modifications using Mass Spectrometry

Mass spectrometry;histone;post-translational modification;efavirenz;cytochrome P450;not listed

Hersman, ElisabethBumpus, Namandje N. ;

Johns Hopkins University

关键词: Mass spectrometry;    histone;    post-translational modification;    efavirenz;    cytochrome P450;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/36977/HERSMAN-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Mouse models are widely used in pharmacology, yet the expression profile of the murine drug metabolizing enzymes has only begun to be characterized at the protein level. We developed a quantitative, high-throughput mass spectrometric method to measure the protein expression of the cytochromes P450 (Cyps) in tissue lysates isolated from Balb/c mice. Global mass spectrometry-based proteomics revealed 27 proteins belonging to Cyp subfamilies 1a, 2a, 2b, 2c, 2d, 2e, 2f, 2j, 2u, 3a, 4a, 4b, 4f, and 4v were readily detectable in Balb/c mouse tissue. Using this protein list, a selected reaction monitoring mass spectrometric screen was developed to quantify expression of these 27 proteins. The screen was applied to mouse liver microsomes and tissue lysates of kidney, lung, intestine, heart and brain from mixed sex fetuses; 3-4 weeks, 9-10 weeks, and 8-10 months of age male and female mice; and pregnant mice. All 27 proteins were identified in liver microsomes. Cyps were identified in all lysates, including 10 in kidney, 15 in lung, 8 in intestine, 6 in heart, and 4 in brain. Liver microsomes differed in expression with sex, age, and pregnancy. Cyps 1a2, 2c67, and 4a12a appeared to be more abundant from male mouse livers of all ages. Hepatic expression of Cyp2b9 was more abundant in 3-4 week old mice in comparison to mice of other ages, and it was the only enzyme identified in higher abundance in pregnant mouse microsomes in comparison to the age matched females. Sexually dimorphic expression appeared exclusively in kidney for Cyps 2b9, 2d26, 2e1, and 4b1. Microsome activity experiments evaluated the mouse liver microsome samples for Cyp activity against the HIV maintenance drug, efavirenz, revealing differences in metabolite formation with age, sex, and pregnancy status. Collectively, these data provide a foundation for the use of mice as a preclinical model for understanding Cyp pharmacology and contributes to a wider understanding of mouse xenobiotic metabolism.In a second set of experiments, a derivatization technique of unmodified lysine residues on histones is used in combination with high performance mass spectrometry to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatized. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.