Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

Abstract

A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.

High‐Throughput Measurement of Binding Kinetics by mRNA Display and Next‐Generation Sequencing

“…Owing to this effect, there is a great need for methods that evaluate the affinity of a ligand for its target in a high‐throughput manner. Advances in the field have increased the throughput of K d measurements by using radioactivity, SPR or fluorescence microarrays, and ELISA assays . However, all of these methods require individually expressed and purified ligands, greatly reducing their throughput.…”

High‐Throughput Measurement of Binding Kinetics by mRNA Display and Next‐Generation Sequencing

“…[4] The challenge, increasingly, is to rank the molecules based on desirable properties, chiefly, their affinity for their targets. [5] …”

“…Advances in the field have increased the throughput of K d measurements using radioactivity, [5a] SPR or fluorescent microarrays, [6] and ELISA assays. [5b] However, all these methods require individually expressed and purified ligands, greatly reducing their throughput. Measuring the K d for thousands of potential ligands simultaneously has not yet been achieved.…”

General, Label-Free Method for Determining Kd and Ligand Concentration Simultaneously

“…Many factors can complicate accurate measurement of ligand concentration, such as unknown expression levels, or unknown fraction of functional/correctly folded ligand, or the desire to use crude, unpurified samples for highest throughput. 19 This prerequisite is one issue that makes highthroughput K d screens for ligands difficult, time-consuming, and infeasible on a proteomic scale.…”