期刊
JOURNAL OF PROTEOME RESEARCH
卷 10, 期 1, 页码 241-248出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr1008898
关键词
CYP27A1; CYP46A1; absolute quantification; multiple reaction monitoring; mass spectrometry; membrane protein; human brain; human retina
资金
- National Institutes of Health [EY018383, AG024336]
- Visual Sciences Training Program [T32 EY07157]
- Research to Prevent Blindness Foundation
- NATIONAL EYE INSTITUTE [T32EY007157, R01EY018383] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [K02AG024336] Funding Source: NIH RePORTER
Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using N-15-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.
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