Tracking a New Cell-Penetrating (W/R) Nonapeptide, through an Enzyme-Stable Mass Spectrometry Reporter Tag
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- 作者：Diane Delaroche ; Baptiste Aussedat ; Soline Aubry ; Gé ; rard Chassaing ; Fabienne Burlina ; Gilles Clodic ; Gé ; rard Bolbach ; Solange Lavielle ; Sandrine Sagan
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：March 1, 2007
- 年：2007
- 卷：79
- 期：5
- 页码：1932 - 1938
- 全文大小：611K
- 年卷期：v.79,no.5(March 1, 2007)
- ISSN：1520-6882
文摘
We have designed a mass stable reporter (msr) tag withm/z over 500, trifluoroacetyl(
,-diethyl)Gly-Lys(N
biotin)-(D)Lys-Cys, for the quantification of the uptake and studyof the degradation processes of cell-penetrating peptides(CPP), by matrix assisted laser desorption/ionizationtime-of-flight (MALDI-TOF) mass spectrometry. This tagwas found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis basedmethod, an accurate tracking of a new CPP and of itsdegradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR-NH2) enters chinesehamster ovary (CHO) K1 cells with a kinetic reaching asteady state after 30-60 min of incubation. This plateauwas stable for 4 h and decreased slowly afterward. (2) Thepeptide msr(W/R) nonapeptide was not cytotoxic over48 h incubation with CHO cells. (3) After 1 h incubation,the msr(W/R) nonapeptide accumulated with a 3-foldhigher concentration than the extracellularly added concentration (7.5 
M). (4) The intracellular quantificationwas accurate with less than 3% of the quantified peptidebeing potentially membrane-bound. (5) There was noleakage of the full-length CPP outside the cells. And,finally, (6) analysis of the degradation process of this newCPP suggests that the peptide did not traffick to lyso-somes.
,-diethyl)Gly-Lys(Nbiotin)-(D)Lys-Cys, for the quantification of the uptake and studyof the degradation processes of cell-penetrating peptides(CPP), by matrix assisted laser desorption/ionizationtime-of-flight (MALDI-TOF) mass spectrometry. This tagwas found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis basedmethod, an accurate tracking of a new CPP and of itsdegradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR-NH2) enters chinesehamster ovary (CHO) K1 cells with a kinetic reaching asteady state after 30-60 min of incubation. This plateauwas stable for 4 h and decreased slowly afterward. (2) Thepeptide msr(W/R) nonapeptide was not cytotoxic over48 h incubation with CHO cells. (3) After 1 h incubation,the msr(W/R) nonapeptide accumulated with a 3-foldhigher concentration than the extracellularly added concentration (7.5 M). (4) The intracellular quantificationwas accurate with less than 3% of the quantified peptidebeing potentially membrane-bound. (5) There was noleakage of the full-length CPP outside the cells. And,finally, (6) analysis of the degradation process of this newCPP suggests that the peptide did not traffick to lyso-somes.