February 15, 2011 (Vol. 31, No. 4)

Allowing Simultaneous Detection, Identification, and Quantification of Protein-Ligand Interactions

Protein-protein interactions are integral to a wide range of biological processes, including hormone, neurotransmitter or drug binding, antigen recognition, and enzyme-substrate interactions. Fundamental to each of these interactions is the recognition by a ligand of a unique binding surface where it binds in a defined way to carry out its function.

Through an understanding of these specific interactions it may be possible to design or discover analogous ligands with altered binding properties and, therefore, to intervene in the biochemical pathway in a highly specific manner. Thus, it is extremely important to delineate the structural characteristics of the bound species in order to develop a model of the binding interaction.

Once one or more protein-protein interactions have been identified, it is desirable to investigate the interaction(s) in order to obtain a mechanistic understanding of the proteins at the molecular level; understand the interactions functional significance in vivo; and develop methods for specifically disrupting or perturbing the interaction in vivo.

A number of affinity-based methods have recently been established for the detection and quantification of biomolecular interactions. Methods widely used for characterization of protein-ligand interactions include immunoaffinity-analysis by ELISA and Western blot, isothermal titration calorimetry, and surface plasmon resonance. Most recently, the SAW (surface acoustic wave) biosensor has become increasingly important for the study of biomacromolecular interactions due to its high detection sensitivity in dilute solutions and ability to use crude biological samples.

The sam®5 biosensor (SAW Instruments) is a chip-based bioaffinity system for real-time, label-free detection of affinity interactions. Unlike SPR which is an optical method, SAW is based on the conversion of a high-frequency signal into a surface acoustic wave due to the inverse piezoelectric effect. The velocity of the SAW is sensitive to changes in mass loading and viscosity, causing shifts in the signal’s amplitude and/or phase, which enable high-sensitivity detection, unlike most other biosensors that are equipped with a readout unit containing a microfluidic cell. Thus, interactions on the gold-coated chip surface can be observed at near-physiological conditions.

A newly developed method enables both identification and quantification of bioaffinity interactions of biopolymers and was applied for characterization of interaction between immobilized Substance P or Melittin, with Calmodulin. Melittin and Substance P were manually covalently bound to the five sensor elements of the gold Sensor Chip surface, which was prefunctionalized with a thiol-linker. Affinity binding experiments were started after changing to a physiological pH by the injection of Calmodulin. The interface used for online coupling of sam®5 with ESI-MS includes a six-port switching valve unit and reverse phase C4-microguard-column for desalting and concentration of dissociated ligand sample (Figure 1).

Figure 1. The on-line SAW-ESI-MS combination using a sam®5 biosensor and a six-port-valve microcolumn interface. (A) sam®5 curve of affinity binding and total dissociation (elution) of the ligand; (B) Interface used for sample concentration and desalting of the ligand; and (C) ESI-MS spectrum of the ligand eluted from the sensor chip.

Melittin shows an approximately 2.5 times higher affinity binding to Calmodulin as compared with Substance P to Calmodulin. Determination of kinetic data and dissociation constants was performed by a number of Calmodulin injections with increasing concentration alternating with surface regeneration steps, and provides for both systems KD values in low nanomolar range (Figure 2).

The new method has shown to be applicable for several systems and enables the direct connection of KD analysis for in vitro biomarkers with quantitation and structural characterization by mass spectrometry. The method has also been further applied to other clinically important systems, e.g., human β-amyloid peptides (Alzheimer disease) and human α-synclein (Parkinson disease).

Figure 2. (A) sam®5 binding curves of Calmodulin obtained using on-line SAW-ESI-MS. (B) Mass spectrometric identification of eluted Calmodulin.

Mihaela Stumbaum is a research assistant at the University of Konstanz, Thomas Gronewold ([email protected]) is an application scientist at SAW, and Michael Przybylski is a professor in the laboratory of analytical chemistry and biopolymer structure analysis, department of chemistry at University of Konstanz.

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