Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins

Published

June 19, 2012

Author(s)

Illarion V. Turko, Meiyao Wang, Gun-Young Heo, Saida Omarova, Irina A. Pikuleva

Abstract

Quantitative analysis of low-abundance membrane proteins remains a challenge and requires further optimization of analytical methods developed for their soluble counterparts. Use of stable isotope-labeled full-length proteins as an internal standard in multiple reaction monitoring (MRM) analysis makes possible pre-fractionation of the target proteins and quantification of those low-abundance proteins, which cannot be reached without biological sample enrichment. In terms of membrane proteins, this benefit can be used if a sample processing workflow will allow complete solubilization of membrane proteins under conditions compatible with the following trypsinolysis and mass spectrometry analysis. We report on the optimization of solubilization conditions of various membrane-bound cytochrome P450s (CYPs) and their electron transferring protein partners, cytochrome P450 reductase (CPR), ferredoxin reductase (FdR), and ferredoxin (Fdx), all important for cholesterol elimination from different organs. The developed protocol enabled MRM quantification of both weakly- (CPR and FdR) and tightly-bound (CYP7B1, CYP11A1, CYP27A1, and CYP46A1) membrane proteins. Measurements were performed on three human tissues (temporal lobe of the brain, retina and retinal pigment epithelium) from multiple donors and the biological implications of our quantitative measurements are discussed.

Citation

Analytical Chemistry

Volume

84

Issue

12

Pub Type

Journals

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DOI Link

Keywords

CYP, P450, absolute quantification, multiple reaction monitoring, mass spectrometry, membrane protein, human brain, human retina, human retinal pigment epithelium.

Citation

Turko, I. , Wang, M. , Heo, G. , Omarova, S. and Pikuleva, I. (2012), Sample prefractionation for mass spectrometry quantification of low-abundance membrane proteins, Analytical Chemistry, [online], https://doi.org/10.1021/ac300587v (Accessed December 14, 2024)