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

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 is
measured by mass spectrometry. We demonstrate the approach using a
peptide library derived from the Escherichia 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 20 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.