Label-Free, In-Solution Screening of Peptide Libraries for Binding to Protein Targets Using Hydrogen Exchange Mass Spectrometry

Abstract

There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g. phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange was measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the E. coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal twenty residues of bovine ribonuclease A (S peptide). Human calmodulin and bovine ribonuclease S (RNase S) were screened against the library. Using a novel data analysis workflow we identified the eNOS peptide as the only calmodulin binding peptide and S peptide as the only ribonuclease S binding peptide in the library.

Ultra-large chemical libraries for the discovery of high-affinity peptide binders

“…OBOC libraries can easily incorporate more varied positions, but OBOC screening is technically challenging and typically examines onlỹ 10 6 compounds 24 . Affinity selection-mass spectrometry (AS-MS) [25][26][27][28][29] represents an alternative strategy for target-based discovery of chemically accessed peptide binders. We recently leveraged LC-MS/MS for sequencing individual synthetic peptides present in complex mixtures 30 , and to increase the diversity of synthetic peptide libraries amenable to AS-MS 12 , from~10 to~10 6 .…”

Protein dynamics revealed by hydrogen/deuterium exchange mass spectrometry: Correlation between experiments and simulation

“…In recent years, HDX‐MS has been developed as a tool for quality control of biopharmaceutical molecules, such as mono‐antibodies and antibody‐drug conjugates, which are becoming an essential part of the drug industry . Furthermore, HDX‐MS has been extensively used in drug screening in the field of drug development, probably due to the development of robust automated HDX‐MS platforms.…”

“…Reprinted with permission from McAllister and Konermann 33 control of biopharmaceutical molecules, such as mono-antibodies and antibody-drug conjugates, which are becoming an essential part of the drug industry. 47 Furthermore, HDX-MS has been extensively used in drug screening in the field of drug development, 48 between the dynamics information obtained by MD simulation and HDX, and the combination of HDX-MS with MD simulation can be a powerful approach to interpret some complicated results and facilitate protein research. 28…”

Time, space, and disorder in the expanding proteome universe

“…deuterium to hydrogen) exchange to map peptidome‐wide peptide–protein interactions. This study highlights the fundamental possibility of exploiting atomic changes to map protein interactions on a global scale . Using HDX technologies on whole cells for cellular structural studies is a desirable extension of the method, however a significant hurdle is the need to minimize back‐exchange during necessary processing steps such as cell lysis and protein digestion prior to bottom‐up LC‐MS/MS analysis.…”