Optimizing the Conditions of a Multiple Reaction Monitoring Assay for Membrane Proteins: Quantification of Cytochrome P450 11A1 and Adrenodoxin Reductase in Bovine Adrenal Cortex and Retina

Abstract

Approximately 30% of naturally occurring proteins are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is traditionally understudied due to limitations of the available analytical tools. To facilitate the analysis of membrane proteins, the analytical methods for their soluble counterparts must be optimized or modified. Multiple reaction monitoring (MRM) assays have proven successful for the absolute quantification of proteins and for profiling protein modifications in cell lysates and human plasma/serum but have found little application in the analysis of membrane proteins. We report on the optimization of sample preparation conditions for the quantification of two membrane proteins, cytochrome P450 11A1 (CYP11A1) and adrenodoxin reductase (AdR). These conditions can be used for the analysis of other membrane proteins. We have demonstrated that membrane proteins that are tightly associated with the membrane, such as CYP11A1, can be quantified in the total tissue membrane pellet obtained after high-speed centrifugation, whereas proteins that are weakly associated with the membrane, such as AdR, must be quantified in the whole tissue homogenate. We have compared quantifications of CYP11A1 using two different detergents, RapiGest SP and sodium cholate, and two different trypsins, sequencing grade modified trypsin and trypsin, type IX-S from porcine pancreas. The measured concentrations in these experiments were similar and encouraged the use of either combination of detergent/trypsin for quantification of other membrane proteins. Overall, the CYP11A1 and AdR quantified in this work ranged from 110 pmol to 10 fmol per milligram of tissue protein.