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Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.

The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase., (Copyright © 2015 Elsevier Inc. All rights reserved.)