title: Selenium as an alternative peptide label - comparison to fluorophore-labelled penetratin.
abstract: In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenM(Se), TAMRA-PenM(Se) and TAMRA-Pen. The labelled peptides were characterized in terms of hydrodynamic radius, secondary structure during peptide-membrane interaction, effect on membrane leakage induction, uptake efficiency in HeLa cells. Furthermore, stability of peptides and identities of degradation products in cell media and cell lysate were evaluated. TAMRA-labelling increased the hydrodynamic radius of Pen and PenM(Se) significantly. Labelling with Se caused no or minimal changes in size, secondary structure and membrane leakage induction in concentration levels relevant for cellular uptake studies. Similar degradation patterns of all labelled peptides were observed in HBSS media; degradation was mainly due to oxidation. Cellular uptake was significantly higher for the TAMRA labelled peptides as compared to Se-labelled Pen. Extensive degradation was observed in media during cellular uptake studies, however, in all cell lysates, primarily the intact peptide (PenM(Se), TAMRA-PenM(Se) or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry. Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels.
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark.
authors.forename: Laura
authors.initials: L
authors.lastname: Hyrup Møller
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark.
authors.forename: Jesper Søborg
authors.initials: JS
authors.lastname: Bahnsen
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark.
authors.forename: Hanne Mørck
authors.initials: HM
authors.lastname: Nielsen
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Østergaard
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark.
authors.forename: Stefan
authors.initials: S
authors.lastname: Stürup
authors.affiliation: Department of Pharmacy, University of Copenhagen, Denmark. Electronic address: bente.gammelgaard@sund.ku.dk.
authors.forename: Bente
authors.initials: B
authors.lastname: Gammelgaard
mesh_terms: D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000494:pharmacology; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000494:pharmacology; D002784:Cholesterol / Q000737:chemistry; D002942:Circular Dichroism; D004355:Drug Stability; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000737:chemistry* / Q000494:pharmacology; D012643:Selenium / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D012235:Rhodamines; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D002784:Cholesterol; C414409:penetratin; D012643:Selenium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: Cell-penetrating peptides; Labelling techniques; Peptide quantification