Fig. 7: ClickZip-tagged peptide use and quantification in cell cultures.

A Schematic depicting the use of ClickZip chelates as mass tags for quantitative analysis of labelled peptides. The key aspect is that the tag is quantitatively released into solution from both the peptide and the cells by total acidic hydrolysis and quantified in the form of an intact metal Ln^III chelate. B Four tagged model peptide conjugates were prepared based on two cell-penetrating hexapeptides (different sequences of L-arginine, D-arginine and NO₂-protected L-arginine) labelled with Lu- or Tm-containing ClickZip tags. C Pairs of conjugates were internalized to CCRF-CEM cells (in triplicates), followed by total acidic hydrolysis (as shown in (A)) before tag quantification. D LC-MS (liquid chromatography—mass spectrometry, single quadrupole) quantification of the intact ClickZip tags in cell lysate demonstrates on paired comparisons that internalization of the conjugates is a function of the peptide sequence, not of the metal in the ClickZip tags. Additional details on the method of analysis are in Supplementary Fig. 25. E The same samples from (D) were analysed with ICP-OES (inductively coupled plasma—optical emission spectroscopy) for direct detection of Lu and Tm, showing excellent agreement with results from panel D and demonstrating that quantification of ClickZip tags with commonplace LC-MS instrumentation provides results equal to more specialized and less common techniques. For (D) and (E), the data are presented as the mean ± SD (N = 3, measurements on independent cell lysates from 10⁶ cells each). Source data available in Supplementary Data 2.