G protein-coupled receptor quantification using peptide group-specific enrichment combined with internal peptide standard reporter calibration

Abstract

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules.

Keywords: 5-HT; 5-hydroxytryptamine (serotonin); ESI; G protein-coupled receptor; G-protein coupled receptor; GPCR; IPA; Immunoaffinity-MS; LC–MS; LLOD; LLOQ; MALDI; MFI; Membrane protein; PE; RSD; RT; S/N; TXP; Triple X proteomics; electrospray ionization; iTRAQ®; isobaric tag for relative and absolute quantification; isopropyl alcohol; liquid chromatography–mass spectrometry; lower limit of detection; lower limit of quantification; mAChR; matrix assisted laser desorption ionization; mean fluorescence intensity; muscarinic acetylcholine receptor; phycoerythrin; relative standard deviation; retention time; signal-to-noise.