Sample Prefractionation for Mass Spectrometry Quantification of Low-Abundance Membrane Proteins

Abstract

Use 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.