High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale

Abstract

Summary Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.

Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype

“…To identify the core binding motif, we characterized the importance of each residue of P2 for binding to HBc by µSPOT peptide microarrays [ 47 ]. Probing C -terminal and N -terminal truncated peptide variants revealed “SLLGRM” as the core-binding motif ( Figure 6 a).…”

Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change

“…To assess the peptide purity after synthesis, which may vary depending on peptide length and sequence, we recommend conducting liquid chromatography coupled to mass spectrometry (LC-MS) measurements of control peptides ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) (see 18). Alternatively, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry can be performed to determine peptide purity.…”

“…Affinity data obtained with peptides of largely differing length can be expected to be biased by variations in amounts and purities. In this line, we recommend an upper limit of 30 aas for affinity determination, since a commonly observed coupling efficiency of 93%–98% corresponds to a peptide purity of 11%–55% ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) . Generally, representative quality controls should always be included in any peptide library to assess the peptide purity (see Quality Control by LC-MS).…”

“…Their subsequent use for high-throughput protein interaction screening as well as affinity determination in solution provides binding data for thousands of unique peptides with a turnover of 1 to 2 weeks, thereby facilitating in vitro assessment and development of high-affinity binders. For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020) …”

“…For complete details on the use and execution of this protocol, please refer to Schulte et al., (2020) …”