Measurements of the Intracellular Stability of CPPs

  • Ivo R. Ruttekolk
  • Wouter P. R. Verdurmen
  • Yi-Da Chung
  • Roland Brock
Part of the Methods in Molecular Biology book series (MIMB, volume 683)


Nowadays, the analysis of the uptake and intracellular distribution of cell-penetrating peptides mostly relies on fluorescence microscopy, using fluorescently labeled CPP analogs. However, fluorescence microscopy does not reveal to which degree fluorescence reflects the intact peptide or only breakdown products. Here, we introduce fluorescence correlation spectroscopy (FCS) as a powerful method to address peptide stability in cells and cell lysates. Measurements in lysates of cells incubated with peptide yield information on degradation of the total cellular peptide content. In combination with protease inhibitors, such measurements enable conclusions on trafficking pathways. Intracellular FCS measurements provide direct information on peptide degradation and association with cellular structures in intact cells.

Key words

Cell-penetrating peptide Fluorescence correlation spectroscopy Proteolytic stability Fluorescence Microscopy Peptide degradation 



The authors acknowledge financial support from the Volkswagen-Foundation (Nachwuchsgruppen an Universitäten, I/77 472) and from the Radboud University Nijmegen Medical Centre to WPRV.


  1. 1.
    Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides, Traffic. 8, 848–866.CrossRefPubMedGoogle Scholar
  2. 2.
    Fotin-Mleczek, M., Welte, S., Mader, O., Duchardt, F., Fischer, R., Hufnagel, H., Scheurich, P., and Brock, R. (2005) Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization, J. Cell Sci. 118, 3339–3351.CrossRefPubMedGoogle Scholar
  3. 3.
    Herbig, M. E., Weller, K. M., and Merkle, H. P. (2007) Reviewing biophysical and cell biological methodologies in cell-penetrating peptide (CPP) research, Crit. Rev. Ther. Drug Carrier Syst. 24, 203–255.PubMedGoogle Scholar
  4. 4.
    Henriques, S. T., Melo, M. N., and Castanho, M. A. (2007) How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo (Review), Mol. Membr. Biol. 24, 173–184.CrossRefPubMedGoogle Scholar
  5. 5.
    Vives, E., Richard, J. P., Rispal, C., and Lebleu, B. (2003) TAT peptide internalization: seeking the mechanism of entry, Curr. Protein Pept. Sci. 4, 125–132.CrossRefPubMedGoogle Scholar
  6. 6.
    Elmquist, A. and Langel, Ü. (2003) In vitro uptake and stability study of pVEC and its all-D analog, Biol. Chem. 384, 387–393.CrossRefPubMedGoogle Scholar
  7. 7.
    Palm, C., Jayamanne, M., Kjellander, M., and Hällbrink, M. (2007) Peptide degradation is a critical determinant for cell-penetrating peptide uptake, Biochim. Biophys. Acta 1768, 1769–1776.CrossRefPubMedGoogle Scholar
  8. 8.
    Trehin, R., Nielsen, H. M., Jahnke, H. G., Krauss, U., Beck-Sickinger, A. G., and Merkle, H. P. (2004) Metabolic cleavage of cell-penetrating peptides in contact with epithelial models: human calcitonin (hCT)-derived peptides, Tat(47–57) and penetratin(43–58), Biochem. J. 382, 945–956.CrossRefPubMedGoogle Scholar
  9. 9.
    Fischer, R., Bachle, D., Fotin-Mleczek, M., Jung, G., Kalbacher, H., and Brock, R. (2006) A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway, ChemBioChem. 7, 1428–1434.CrossRefPubMedGoogle Scholar
  10. 10.
    Pillay, C. S., Elliott, E., and Dennison, C. (2002) Endolysosomal proteolysis and its regulation, Biochem. J. 363, 417–429.CrossRefPubMedGoogle Scholar
  11. 11.
    Räägel, H., Säälik, P., Hansen, M., Langel, U., and Pooga, M. (2009) CPP-protein constructs induce a population of non-acidic vesicles during trafficking through endo-lysosomal pathway, J. Control Release 139, 108–117.CrossRefPubMedGoogle Scholar
  12. 12.
    Tunnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., and Cardoso, M. C. (2006) Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells, FASEB J. 20, 1775–1784.CrossRefPubMedGoogle Scholar
  13. 13.
    Burlina, F., Sagan, S., Bolbach, G., and Chassaing, G. (2005) Quantification of the cellular uptake of cell-penetrating peptides by MALDI-TOF mass spectrometry, Angew. Chem. Int. Ed. Engl. 44, 4244–4247.CrossRefPubMedGoogle Scholar
  14. 14.
    Burlina, F., Sagan, S., Bolbach, G., and Chassaing, G. (2006) A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry, Nat. Protoc. 1, 200–205.CrossRefPubMedGoogle Scholar
  15. 15.
    Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development, Biochemistry 37, 6235–6239.CrossRefPubMedGoogle Scholar
  16. 16.
    EL Andaloussi, S., Guterstam, P., and Langel, Ü. (2007) Assessing the delivery efficacy and internalization route of cell-penetrating peptides, Nat. Protoc. 2, 2043–2047.CrossRefGoogle Scholar
  17. 17.
    Rigler, R., Mets, U., Widengren, J., and Kask, P. (1993) Fluorescence correlation spectroscopy with high count rate and low-background – analysis of translational diffusion, Eur. Biophys. J. Biophys. Lett. 22, 169–175.Google Scholar
  18. 18.
    Bacia, K. and Schwille, P. (2007) Fluorescence correlation spectroscopy, Methods Mol. Biol. 398, 73–84.CrossRefPubMedGoogle Scholar
  19. 19.
    Waizenegger, T., Fischer, R., and Brock, R. (2002) Intracellular concentration measurements in adherent cells: a comparison of import efficiencies of cell-permeable peptides, Biol. Chem. 383, 291–299.CrossRefPubMedGoogle Scholar
  20. 20.
    Rich, D. H., Bernatowicz, M. S., Agarwal, N. S., Kawai, M., Salituro, F. G., and Schmidt, P. G. (1985) Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition, Biochemistry 24, 3165–3173.CrossRefPubMedGoogle Scholar
  21. 21.
    Tamai, M., Matsumoto, K., Omura, S., Koyama, I., Ozawa, Y., and Hanada, K. (1986) In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64, J. Pharmacobiodyn. 9, 672–677.PubMedGoogle Scholar
  22. 22.
    Koppel, D. E. (1974) Statistical accuracy in fluorescence correlation spectroscopy, Phys. Rev. A 10, 1938–1945.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ivo R. Ruttekolk
    • 1
  • Wouter P. R. Verdurmen
    • 1
  • Yi-Da Chung
    • 1
  • Roland Brock
    • 1
  1. 1.Department of Biochemistry, Nijmegen Centre for Molecular Life SciencesRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

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