title: A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import.
abstract: Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the determination of the intracellular concentrations for each construct individually.
authors.affiliation: Group of Genomics and Proteomics, Center for Bioinformatics Tübingen, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Thomas
authors.initials: T
authors.lastname: Waizenegger
authors.forename: Karsten
authors.initials: K
authors.lastname: Köhler
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D000595:Amino Acid Sequence; D051943:Antennapedia Homeodomain Protein; D002352:Carrier Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D057846:Cell-Penetrating Peptides; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D009687:Nuclear Proteins; D010455:Peptides / Q000378:metabolism*; D012235:Rhodamines; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D014407:Tumor Cells, Cultured
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D051943:Antennapedia Homeodomain Protein; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; D026901:Membrane Transport Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012235:Rhodamines; D014157:Transcription Factors; C024098:6-carboxyfluorescein; C414409:penetratin