Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides, CPPs, has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences.

Here we demonstrate a method for in-cell penetration selection-mass spectrometry, in-cell PS-MS, to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation.

A representative peptide discovered in the cytosolic extract, Cyto1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer, PMO, delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Cyto1a is composed of D-residues and two non-α-amino acids, is more stable than its all-L isoform, and is less toxic than known CPPs with comparable activity. Pulse-chase and microscopy experiments revealed that while the PMO-Cyto1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus without nonspecifically releasing other endosomal components. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.