Tracking a new cell-penetrating (W/R) nonapeptide, through an enzyme-stable mass spectrometry reporter tag.
We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(alpha,alpha-diethyl)Gly-Lys(Nepsilonbiotin)-(D)Lys-Cys, for the quantification of the uptake and study of the degradation processes of cell-penetrating peptides (CPP), by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This tag was found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis based method, an accurate tracking of a new CPP and of its degradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR-NH2) enters chinese hamster ovary (CHO) K1 cells with a kinetic reaching a steady state after 30-60 min of incubation. This plateau was stable for 4 h and decreased slowly afterward. (2) The peptide msr(W/R) nonapeptide was not cytotoxic over 48 h incubation with CHO cells. (3) After 1 h incubation, the msr(W/R) nonapeptide accumulated with a 3-fold higher concentration than the extracellularly added concentration (7.5 microM). (4) The intracellular quantification was accurate with less than 3% of the quantified peptide being potentially membrane-bound. (5) There was no leakage of the full-length CPP outside the cells. And, finally, (6) analysis of the degradation process of this new CPP suggests that the peptide did not traffick to lysosomes.
Delaroche
Diane
D
Synthèse, Structure et Fonction de Molécules Bioactives (CNRS) and FR 2769, UMR 7613, and Plateforme de Spectrométrie de Masse et Protéomique, Université Pierre et Marie Curie-Paris 6, Paris, France.
Aussedat
Baptiste
B
Aubry
Soline
S
Chassaing
Gérard
G
Burlina
Fabienne
F
Clodic
Gilles
G
Bolbach
Gérard
G
Lavielle
Solange
S
Sagan
Sandrine
S
D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D008247:Lysosomes / Q000201:enzymology; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D021381:Protein Transport; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
C520514:H-RRWWRRWRR-NH2; D009842:Oligopeptides; C520513:trifluoroacetyl(alpha,alpha-diethyl)Gly-Lys(Nepsilonbiotin)-(D)Lys-Cys; D001710:Biotin; D011188:Potassium