Enrichment of Membrane Proteins for Downstream Analysis Using Styrene Maleic Acid Lipid Particles (SMALPs) Extraction

Abstract

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein-protein and protein-lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor-ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

Keywords: Affinity Purification; Drosophila melanogaster; Ligand-Receptor Interaction; Mass Spectrometry; Native Membrane Protein Extraction; Nicotinic Acetylcholine Receptor (nAChR); Styrene Maleic Acid Lipid Particles (SMALPs).

Figure 1.. Overview of the protocol to enrich native plasma membrane proteins complexes and use styrene maleic acid lipid particles (SMALPs) enrichment and mass spectrometric analysis.

A. In this example, D. melanogaster heads are used as a starting material. Cell membranes are first enriched using differential centrifugation. Membrane pellets are used to perform the SMALPs extraction. The plasma membrane–enriched fraction, in this case fraction 4, is mixed with SMA. The resulting nanodiscs containing the target protein of interest, in this case nAChRs, are further enriched using affinity beads coupled to α-BTX. Proteins within the discs are identified by firstly digesting to peptides using trypsin followed by LC–MS/MS. B. Western blot analysis to determine the fraction enriched in the plasma membrane using ATPase alpha 1 as marker. C. Western blot analysis to confirm the presence of the target membrane protein, in this example the fluorescent protein–tagged Dα6 nAChR subunit in enriched nanodiscs.