In Vitro Studies for Detection of CYP27A1 Modifications with Iso[4]levuglandin E2 in Human Retina

Published

February 17, 2011

Author(s)

Illarion Turko, Casey Charvet, Wei-Li Liao, Gun-Young Heo, Laird James, Salomon G. Robert, Irina A. Pikuleva

Abstract

Isolevuglandins are highly reactive products of free radical-induced oxidation of arachidonates that modify free amino groups of proteins into pyrrole-derived adducts. These protein adducts provide a "footprint" of oxidative injury and are potential biomarkers for assessing risk for oxidative stress-associated diseases. Here, we used a multiple reaction monitoring (MRM) assay with 15N-labeled protein internal standards for detection and quantitative analysis of pyrrole, lactam, and hydroxylactam adducts of mitochondrial cytochrome P450, CYP27A1. In vitro modification of CYP27A1 with iso[4]LGE2 revealed a limited set of Lys residues susceptible to modification, including Lys358 and Lys476. Analysis of in vitro modified 15N-CYP27A1 revealed the most intense transitions and their intensity ratios for peptides having modified Lys358 and Lys476. This information was further used to detect the lactam and hydroxylactam adducts of CYP27A1 in human neural retina from a donor with dry age-related macular degeneration. Prior to MRM assay, the retina sample supplemented with modified 15NCYP27A1 was enriched in CYP27A1 content using a whole gel elution technique. The combination of MRM assay using 15N-labeled protein internal standard with enrichment of target protein(s) using whole gel elution can be adapted to analysis of other proteins which may be subject to modification in vivo.

Citation

Analytical Chemistry

Pub Type

Journals

Keywords

CYP27A1, multiple reaction monitoring, retina, oxydative stress

Citation

Turko, I. , Charvet, C. , Liao, W. , Heo, G. , James, L. , Robert, S. and Pikuleva, I. (2011), In Vitro Studies for Detection of CYP27A1 Modifications with Iso[4]levuglandin E₂ in Human Retina, Analytical Chemistry (Accessed May 18, 2024)