Surface plasmon resonance for high‐throughput ligand screening of membrane‐bound proteins

Abstract

Technology based on surface plasmon resonance (SPR) has allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions, from quantitative measurements of binding kinetics and thermodynamics and concentrations in complex samples to epitope analysis. SPR has become the gold standard in industrial and academic settings, in which typically the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, the technology is only beginning to be adapted to the needs of membrane-bound proteins. Including G protein-coupled receptors (GPCR), ion channels and other growth, immune and cellular receptors, these proteins are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcore™, have been adapted for membrane protein analysis by building supported lipid layers or vesicle capture on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss successes with current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of GPCR ligands, biomarker discovery involving membrane bound proteins and basic cellular biology.

Metabolites as global regulators: A new view of protein regulation

“…Similarly, steric hindrance to molecular interactions may also result from attachment of proteins onto the stationary matrix and exposure to cytosolic extracts containing metabolites [2,19,20]. To date several approaches have used these types of strategies, such as protein microarrays [2,20], small molecule arrays [7,18], surface plasmon resonance [21], and nuclear magnetic resonance [22]. These in vitro interactions can be affected by factors that include ion strength, solvent, redox, etc., which may not be physiological in vivo.…”

Approaches to Analyze Protein-Protein Interactions of Membrane Proteins

“…Because membrane proteins cannot be bound to chip surface as efficient as soluble proteins, specific chips have been designed that allow the capture of proteoliposomes (Maynard et al 2009). A detailed protocol for this approach is given by (Hodnik and Anderluh 2010).…”

“…Technologies that allow the analysis of membrane proteins directly on a chip are still in develop (Maynard et al 2009). Nevertheless SPR can already be utilized to analyze PPI between a membrane protein and a soluble protein.…”

Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells

“…Currently, the most widespread microfluidic technologies used for cell surface analysis are miniaturized patch clamp systems, 10,11 fluorescence-activated cell sorting (FACS), 12 and surface plasmon resonance systems. 13,14 These microfluidic systems are very good for investigating known interactions of peptides and proteins with cells, but are less suited for use in a discovery mode.…”