Tracking a New Cell-Penetrating (W/R) Nonapeptide, through an Enzyme-Stable Mass Spectrometry Reporter Tag

We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(α,α-diethyl)Gly-Lys(Nεbiotin)-(D)Lys-Cys, for the quantification of the uptake and study of the degradation processes of cell-penetrating peptides (CPP), by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This tag was found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis based method, an accurate tracking of a new CPP and of its degradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR−NH2) enters chinese hamster ovary (CHO) K1 cells with a kinetic reaching a steady state after 30−60 min of incubation. This plateau was stable for 4 h and decreased slowly afterward. (2) The peptide msr(W/R) nonapeptide was not cytotoxic over 48 h incubation with CHO cells. (3) After 1 h incubation, the msr(W/R) nonapeptide accumulated with a 3-fold higher concentration than the extracellularly added concentration (7.5 μM). (4) The intracellular quantification was accurate with less than 3% of the quantified peptide being potentially membrane-bound. (5) There was no leakage of the full-length CPP outside the cells. And, finally, (6) analysis of the degradation process of this new CPP suggests that the peptide did not traffick to lyso-somes.