Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Published

January 7, 2011

Author(s)

Wei-Li Liao, Gun-Young Heo, Nathan G. Dodder, Rachel Reem, Natalia Mast, Suber Huang, Pier L. DiPatre, Illarion Turko, Irina A. Pikuleva

Abstract

Improper cholesterol metabolism in the brain causes neurological diseases. Factors affecting cholesterol homeostasis includes cholesterol hydroxylation reactions, which are regulated by cytochrome P450 enzymes. One approach to estimate the total enzymatic activity of the P450s is to measure the absolute protein concentration in the tissue. An optimized workflow, specifically developed for the quantification of membrane proteins, was used to measure the amount of three P450s, CYP7A1, CYP27A1, and CYP46A1, in the temporal lobe of four individual human brains. The quantification was performed using LC-MS/MS in MRM mode, in combination with nitrogen-15 labeled intact protein standards. We determined the absolute concentration of CYP27A1 and CYP46A1 in human brain; the concentration of CYP7A1 was less than the detection limit of 0.02 pmol/mg total protein. The average CYP27A1 expression level was around 0.1 pmol/mg total protein. The CYP27A1 expression level was identical between the gray and the white brain matter; in contrast the concentration of CYP46A1 was around 1.6-fold higher in the gray matter. The biological significance of this difference requires further investigation. This membrane protein quantification method was reproducible, with CVs from 5% to 25%; therefore, this approach is potentially capable of robust measurements in a clinical setting. The method described here can be expanded to include the quantification of other membrane proteins, for a more complete understanding of the role of P450s in human brain cholesterol homeostasis.

Citation

Journal of Proteome Research

Volume

10

Issue

1

Pub Type

Journals

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DOI Link

Keywords

cytochrome P450, absolute quantification, multiple reaction monitoring, mass spectrometry, membrane protein, human brain

Citation

Liao, W. , Heo, G. , Dodder, N. , Reem, R. , Mast, N. , Huang, S. , DiPatre, P. , Turko, I. and Pikuleva, I. (2011), Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain, Journal of Proteome Research, [online], https://doi.org/10.1021/pr1008898 (Accessed October 5, 2024)