Peptide Microarray-Based Protein Interaction Studies Across Affinity Ranges: Enzyme Stalling, Cross-Linking, Depletion, and Neutralization.

While an ever-increasing number of protein-protein interactions were studied by peptide microarrays with great success, array-based investigations of transiently binding proteins, such as HDACs, and precise binding quantification, remained challenging. Here, we present an updated protocol for the preparation and use of peptide microarrays including the necessary adjustments for simple semi-quantitative and precise measurements across affinity ranges. This procedure describes the mass spectrometric controlled preparation of peptide microarrays in μSPOT format, and their application in binding profiling of recombinant, as well as endogenous, native proteins. We further highlight how cross-linking, blocking, and enzyme stalling can be leveraged to enhance sensitivity and describe how in situ on-chip binding neutralization can enhance the predictive value and robustness of the binding readout. Finally, we included examples for the integration of precise biophysical binding readouts that complement the traditional array-based binding assays.