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Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry
Authors:Baptiste Aussedat
Affiliation:Université Pierre et Marie Curie-Paris6, boite 45, 4 place Jussieu, 75252 Paris cedex 05, France CNRS UMR 7613, Synthèse, Structure et Fonction de Molécules Bioactives, FR2769, France
Abstract:We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.
Keywords:Acm, acetamidomethyl   Boc, tert-butoxycarbonyl   BSA, bovine serumalbumin   CHCA, α-cyano-4-hydroxycinnamic acid   CHO, Chinese hamster ovary   CPP, cell-penetrating peptide   H-CPP, non-deuterated cell-penetrating peptide   D-CPP, deuterated cell-penetrating peptide   2Cl-Cbz, 2-chlorobenzyloxycarbonyl   DCC, dicyclohexylcarbodiimide   DMEM, Dubelcco's modified eagle medium   DIEA, diisopropylethylamine   DMF, N, N-dimethylformamide   DTT, dithiotreitol   EDTA, ethylenediamine tetraacetic acid   HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide   HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide   HOAt, 1-hydroxy-7-aza-benzotriazole   HOBt, 1-hydroxybenzotriazole   HPLC, high performance liquid chromatography   MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry   MBHA-PS, 4-methylbenzhydrylamine polystyrene   4-MeOBzl, p-methoxybenzyl   Npys, 3-nitro-2-pyridinesulphenyl   PyAOP, 7-Azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate   PKC, protein kinase C   PKCi, proteins kinase C inhibitor   PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa-fluorophosphate   SEM, standard error of the mean   TFA, trifluoroacetamide   TFFH, tetramethylfluoroformamidinium hexafluorophosphate   THF, tetrahydrofuran
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