ac300587v_si_001.pdf (463 kB)
Sample Prefractionation for Mass Spectrometry Quantification of Low-Abundance Membrane Proteins
journal contribution
posted on 2012-06-19, 00:00 authored by Meiyao Wang, Gun-Young Heo, Saida Omarova, Irina A. Pikuleva, Illarion V. TurkoUse of stable isotope-labeled full-length proteins as
an internal standard prior to multiple reaction monitoring (MRM) analysis
enables prefractionation 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 allows entire solubilization
of membrane proteins. We have developed a universal workflow for sample
processing and enrichment by optimizing washing and solubilization
conditions and implementing sample fractionation by Whole Gel Eluter.
The optimized protocol was applied to various membrane-bound cytochromes
P450 (CYPs) and their electron transferring protein partners, cytochrome
P450 reductase (CPR), ferredoxin reductase (FdR), and ferredoxin (Fdx),
all important proteins for cholesterol elimination from different
organs. Both, weakly associated (CPR and FdR) and tightly associated
(CYP7B1, CYP11A1, CYP27A1, and CYP46A1) membrane proteins were quantified.
Measurements were performed on three human tissues (temporal lobe
of the brain, retina, and retinal pigment epithelium) obtained from
multiple donors. The biological implications of our quantitative measurements
are also discussed.