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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. [ABSTRACT FROM AUTHOR]

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E2 (iso[4]LGE2) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE2 adduction. CYP27A1 exhibited only three Lys residues, Lys134, Lys358, and Lys476, that readily interact with iso[4]LGE2 in vitro. Such selective modification enabled the generation of an internal standard, 15N-labeled CYP27A1 modified with iso[4]LGE2, for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK358(-C20H26O3)ETLR in the retinal sample were observed that co-eluted with the corresponding two 15N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes. [ABSTRACT FROM AUTHOR]