Advanced mass spectrometry-based strategies for the isolation and characterization of G protein-coupled estrogen receptor 1(GPR)

Abstract

Estrogen signaling plays a vital role in breast, ovarian and endometrial cancers. The actions of estrogen are mainly mediated by classical estrogen receptors, ERα and ERβ that belongs to the nuclear receptor superfamily. In recent years, a class of membrane-associated estrogen receptors are found to mimic the functions of classical ERs, including genomic as well as non-genomic signaling. These non-genomic signaling events include pathways that are usually thought of as arising from transmembrane growth factor receptors and G protein-coupled receptors (GPCRs). GPCRs belong to a superfamily of cell surface signaling proteins. GPCRs represent the most significant family of validated pharmacological targets in medical biology. A member of the GPCR family, named GPER, mediates rapid biological responses to estrogen in diverse normal and cancer cells, as well as transformed cell types. The identification and characterization of GPER will lead to understand the mechanisms underlying complex biological pathways and identify potentially new drug targets. Here, we proposed a novel gel-free method to isolate and enrich GPER from crude lysate using home-made hydroxyapatite column (HTP). The HTP eluate was subjected to cellulose acetate (CA) filteration, followed by on-membrane protein digestion with different proteases and analyzed by MALDI MS. GPER was identified by peptide mass fingerprinting (PMF) after intensive data analysis. Sequence analysis reports 3 potential N-glycosylation in GPER. We manually validated 2 out of 3 glycosylation sites in GPER from the obtained MS/MS data and also validated the glycan moieties predicted by Glycomod. This approach is the first of its kind to identify GPER and characterize post-translational modifications (PTMs) by MS-based proteomic analysis. The proposed method is simple, robust and unique with great reproducibility. Finally, we designed and synthesized polymer nanoparticles (NPs) in an effort to capture GPER with high affinity and selectivity from crude lysate. PNIPAm-based NPs were synthesized by a free radical precipitation polymerization method with no control over the functional monomer sequence. The NP binding affinity was evaluated against both truncated-GPER (short peptide epitopes) and GPER (whole protein). As the NPs were designed with complementary functionality against the peptides/protein, the NPspeptide/ protein binding will be through multipoint interactions. The initial qualitative results obtained by immunoblotting analysis revealed interesting hints on GPER’s competitive affinity towards NPs when probed against multiple antibodies. We anticipate to use this strategy as a sample purification step prior to MS-based proteomic analysis