Nonapeptide, through an Enzyme-Stable Mass Spectrometry Reporter

Gilles Clodic,‡ Ge´ rard Bolbach,†,‡ Solange Lavielle,† and Sandrine Sagan*,†. “Synthe`se, Structure et Fonction de Molécules Bioactives...
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Anal. Chem. 2007, 79, 1932-1938

Tracking a New Cell-Penetrating (W/R) Nonapeptide, through an Enzyme-Stable Mass Spectrometry Reporter Tag Diane Delaroche,*,† Baptiste Aussedat,† Soline Aubry,† Ge´rard Chassaing,† Fabienne Burlina,† Gilles Clodic,‡ Ge´rard Bolbach,†,‡ Solange Lavielle,† and Sandrine Sagan*,†

“Synthe` se, Structure et Fonction de Mole´ cules Bioactives” (CNRS) and FR 2769, UMR 7613, and Plateforme de Spectrome´ trie de Masse et Prote´ omique, Universite´ Pierre et Marie Curie-Paris 6, Case Courrier 182, 4 Place Jussieu, F-75005 Paris, France

We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(r,r-diethyl)Gly-Lys(NEbiotin)(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 µM). (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. Cell-penetrating peptidessCPP (also named protein transduction domainsPTD)1-5sare natural or synthetic peptides identified as cellular membrane crossing molecules or trojan peptides,6 in particular through their potency to vehiculate, to the cytoplasm * To whom correspondence should be addressed. E-mail: [email protected] (S.S.); [email protected] (D.D.) Phone: 33 1 44 27 55 09. Fax: 33 1 44 27 71 50. † UMR 7613. ‡ Plateforme de Spectrome´trie de Masse et Prote´omique. (1) Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1994, 269, 10444-10450. (2) Vives, E.; Brodin, P.; Lebleu, B. J. Biol. Chem 1997, 272, 16010-16017. (3) Joliot, A.; Prochiantz, A. Nat. Cell Biol. 2004, 6, 189-196. (4) Dietz, G. P. H.; Ba¨hr, M. Mol. Cell. Neurosci. 2004, 27, 85-131. (5) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Trends Pharmacol. Sci. 2000, 21, 99-103. (6) Derossi, D.; Chassaing, G.; Prochiantz, A. Trends Cell Biol. 1998, 8, 84-87.

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and nucleus of living cells, various kinds of compounds.4,5 The mechanism of cell entry, energy-dependent or -independent, for these peptides/proteins is controversial. Many processes that allow transduction of one molecule from the extracellular medium to the intracellular one have been stated,7-18 such as the endosomal pathway, macropinocytosis, membrane potential, or inverted micelles. However, it appears now clearly that the mechanism of cell entry strongly depends on the nature and size of the CPP19 and also of the cargo to be transported.20 For example, incorporation on a CPP of a fluorescent probe that might be considered as a hydrophobic cargo and might alter the peptide/membrane interaction has been reported to confer cytotoxic properties to many peptides.21 The consensus point for the studies on CPP is that most of them (if not all) contain basic amino acids, endowing them with a strong positive net charge that is crucial for their entry into cells.22 Although several methods describe cell-uptake CPP quantification (based on radioactivity counting, biotinylation/ cell-ELISA, fluorescence-labeling/spectrophotometer/FACS, resonance energy transfer, HPLC detection, immunodetection, fluorescence correlation microscopy, laser micropipette system, or (7) Berlose, J. P.; Convert, O.; Derossi, D.; Brunissen, A.; Chassaing, G. Eur. J. Biochem. 1996, 242, 372-386. (8) Ziegler, A.; Blatter, X. L.; Seelig, A.; Seelig, J. Biochemistry 2003, 42, 91859194. (9) Rothbard, J. B.; Jessop, T. C.; Wender, P. A. Adv. Drug Deliv. Rev. 2005, 57, 495-504. (10) Henriques, S. T.; Castanho, M. A. Biochemistry 2004, 43, 9716-9724. (11) Terrone, D.; Sang, S. L.; Roudaia, L.; Silvius, J. R. Biochemistry 2003, 42, 13787-13799. (12) Vives, E. J. Mol. Recognit. 2003, 16, 265-271. (13) Vendeville, A.; Rayne, F.; Bonhoure, A.; Bettache, N.; Montcourrier, P.; Beaumelle, B. Mol. Biol. Cell 2004, 15, 2347-2360. (14) Fotin-Mleczek, M.; Welte, S.; Mader, O.; Duchardt, F.; Fischer, R.; Hufnagel, H.; Scheurich, P.; Brock, R. J. Cell Sci. 2005, 118, 3339-3351. (15) Saalik, P.; Elmquist, A.; Hansen, M.; Padari, K.; Saar, K.; Viht, K.; Langel, U.; Pooga, M. Bioconjugate Chem. 2004, 15, 1246-1253. (16) Ross, M. F.; Filipovska, A.; Smith, R. A.; Gait, M. J.; Murphy, M. P. Biochem. J. 2004, 383, 457-468. (17) Wadia, J. S.; Stan, R. V.; Dowdy, S. F. Nat. Med. 2004, 10, 310-315. (18) Wang, W.; El-Deiry, W. S. Trends Biotechnol. 2004, 22, 431-434. (19) Goncalves, E.; Kitas, E.; Seelig, J. Biochemistry 2005, 44, 2692-2702. (20) Maiolo, J. R.; Ferrer, M.; Ottinger, E. A. Biochim. Biophys. Acta 2005, 1712, 161-172. (21) Jones, S. W.; Christison, R.; Bundell, K.; Voyce, C. J.; Brockbank, S. M. V.; Newham, P.; Lindsay, M. A. Brit. J. Pharmacol. 2005, 145, 1093-1102. (22) Jiang, T.; Olson, E. S.; Nguyen, Q. T.; Roy, M.; Jennings, P. A.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17867-17872. 10.1021/ac061108l CCC: $37.00

© 2007 American Chemical Society Published on Web 01/30/2007

cell activity by capillary electrophoresis),23,24 little is known about the catabolism of these peptides inside the cells. One study has addressed this issue using matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis.25 However, degradation of CPP cannot be easily followed by MALDI-TOF MS for ions with mass over charge (m/z) ratio below ∼500. Indeed, from this limiting value, signals of the matrix may interfere with those of interest. Getting information about the catabolism of CPP in cells is crucial to get insight into the entry or uptake mechanism. For that reason, we have designed and synthesized a reporter that could be stable to proteolysis and could be easily detected by MALDI-TOF MS. This tag, trifluoroacetyl-(R,R-diethyl)Gly-Lys(Nbiotin)-(D)Lys-Cys, has been incorporated at the N-terminus of the (W/R) nonapeptide sequence, H-RRWWRRWRR-NH2, giving the putative mass stable reporter (msr) tagged msr(W/R) nonapeptide. The (W/R) nonapeptide sequence is a shorter analogue of the previously reported synthetic CPP, (W/R) hexadecapeptide.6 We report herein that this tag is a reliable tool to study the enzymatic stability, uptake kinetics, and efficiency of the msr(W/R) nonapeptide, as well as its catabolism in cells, using the quantification method by MALDITOF MS previously described.26-28 In addition, we could further demonstrate that this method of quantification is accurate by measuring the remaining peptide bound to the membrane after washing and trypsin treatments. EXPERIMENTAL SECTION Cell Culture. Chinese hamster ovary (CHO) K1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 000 IU/ L), streptomycin (100 000 IU/L), and amphotericin B (1 mg/L) in a humidified atmosphere containing 5% CO2 at 37 °C. Peptide Purification by Streptavidin-Coated Magnetic Beads. Peptides were captured by incubation for 30 min at room temperature (RT) with 100 µg of streptavidin-coated magnetic beads (Dynal-InVitrogen, France). The beads were washed 3 times with 200 µL of 50 mM Tris-HCl buffer, pH 7.4, 0.1% BSA (buffer A) and with H2O (3 × 200 µL, 1 × 100 µL, 1 × 50 µL, and 1 × 5 µL). Proteolysis by Trypsin/Chymotrypsin or Pepsin. These assays have been conducted with 0.1 nmol of peptide in the presence of a trypsin/chymotrypsin mixture (30 µg of each enzyme) for 10 min at 37 °C in 100 mM Na2HCO3, pH 8; or pepsin (40 µg) for 10 min at RT in 10 mM HCl, pH 2. The samples were then diluted with a 50 mM Tris-HCl buffer, pH 7.4, 0.1% BSA before purification by the streptavidin-coated magnetic beads. Proteolysis by Cell Lysates. CHO cells grown to confluence (≈107 cells) in 100 mm culture dishes were washed with 10 mL (23) Chassaing, G.; Sagan, S.; Lequin, O.; Lamazie`re, A.; Ayala-Sanmartin, J.; Trugnan, G.; Bolbach, G.; Burlina, F. In Handbook of Cell-Penetrating Peptides, 2nd edi.; Langel, U ¨ ., Ed.; CRC Press: Boca Raton, FL, 2006; pp 89-107. (24) Lindgren, M.; Ha¨llbrink, M.; Langel, U ¨ . In Cell-Penetrating Peptides Processes and Applications; Langel, U ¨ ., Ed.; CRC Press: Boca Raton, FL, 2002; pp 263-275. (25) Elmquist, A.; Langel, U ¨ . Biol. Chem. 2003, 384, 387-393. (26) Burlina, F.; Sagan, S.; Bolbach, G.; Chassaing, G. Angew. Chem., Int. Ed. 2005, 44, 4244-4247. (27) Burlina, F.; Sagan, S.; Bolbach, G.; Chassaing, G. Nat. Protocols 2006, 1, 200-205. (28) Aussedat, B.; Sagan, S.; Chassaing, G.; Bolbach, G.; Burlina, F. Biochim. Biophys. Acta 2006, 1758, 375-383.

of DMEM, collected, and broken by sonication for 10 min at RT. 1H-msr(W/R) nonapeptide (1 µM) in 2 mL of cell lysates was incubated for 30 min at 37 °C. Samples were heated for 15 min at 100 °C and then centrifuged at 10 000 rpm for 5 min at 4 °C. The pellet was resuspended in 500 µL of 50 mM Tris-HCl buffer, pH 7.4, 0.1% BSA, and 3 mM dithiothreitol (DTT) and centrifuged again at 10000 rpm for 4 min at 4 °C. The supernatants were collected and incubated with 100 µg of streptavidin-coated magnetic beads for 2 h at 4 °C. Uptake Assays. The uptake experiments were performed as previously described.26,27 Briefly, cells for uptake were seeded in 12-well plates at a density of 500 000-1 000 000 cells/well in 2 mL of DMEM with 10% FCS, the day before. The biotinylated 1H-peptide (7.5 µM) in 1 mL of culture medium was incubated with adherent CHO cells (106 cells/well) for 75 min at 37 °C. Purification was achieved with streptavidin-coated beads. Cytotoxicity Assays. Cell suspension (100 µL, 1500 CHO cells/well) were seeded in DMEM plus 10% FCS in 96-well microtiter plate at 37 °C. The peptide (10 µL) was added to the cells to give final concentrations of 0.5, 1, or 10 µM after 24 and 48 h of cell culture. The CCK8 cell counting kit was used according to the supplier (Dojindo Laboratories) and absorbance measured at 450 nm using a microplate reader (FLUOstar OPTIMA, BMG LABTECH) with a reference wavelength at 620 nm. Outflow Assays. After cell uptake, the cells were washed twice with 2 mL of culture medium (DMEM), treated or not with trypsin (0.05% for 2 min). The cells were then incubated again at 37 °C with 1 mL of culture medium for different times (0, 15, 60, and 120 min). The outflow process was measured indirectly by quantifying the peptide in cells. Mass Spectrometry Analysis. Mass characterization of the peptides was performed using a MALDI-TOF/TOF AB 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems) in positive ion reflector mode and delayed extraction. The matrix used in all the experiments was R-cyano-4-hydroxycinnamic acid (HCCA) saturated solution in acetronitrile:0.1% TFA (1:1). The peptides purified on the beads were released with 3 µL of R-cyano4-hydroxycinnamic acid at RT for 10 min, and after beads immobilization with the Dynal magnetic particle concentrator, 0.5 µL of the elution mixture was deposited on the sample holder. The mass spectrometry data analysis was described previously in detail.26,27 Briefly, the quantification of the msr(W/R) nonapeptide was based on the use of an internal standard (deuterated msr(W/ R) nonapeptide), allowing a direct comparison of the quantity vs the ion signal intensities. In all these experiments, ion signal intensities were determined using the peak area of the isotopic pattern. For the experiment of peptide degradation, the ion abundance of each fragment was calculated as the ratio of its ion signal intensity to the total ion signal intensity (fragments plus intact peptide). RESULTS AND DISCUSSION Stability of the Mass Reporter Tag. The trifluoroacetyl-(R,Rdiethyl)Gly-Lys(Nbiotin)-(D)Lys-Cys tag has been synthesized (Supporting Information) and incorporated at the N-terminus of the (W/R) nonapeptide (Figure 1A). This msr(W/R) nonapeptide was first incubated with a 1:1 mixture of trypsin and chymotrypsin for 10 min at 37 °C. MS analysis (not shown) demonstrated that Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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Figure 1. (A) Peptide sequence of the msr(W/R) nonapeptide. The (M + H)+ values of the full-length and degradation products of the peptide are also indicated as observed in spectra from MALDI-TOF analysis. Examples of representative MALDI-TOF mass spectra (external calibration) of B, the full-length 1H-form and 2H-form of msr(W/R) nonapeptides for quantification as reported previously,24 and C (inset), degradation fragments that are observed after cell-uptake of msr(W/R) nonapeptide in CHO K1 cells. Nonattributed peaks are oxidized (biotin) forms of the peptide ions.

the full-length peptide disappeared to lead to only one peptide fragment (with [M + H]+ at m/z 969.46, first isotope). This fragment results from the cleavage of the msr(W/R) nonapeptide after the first N-terminal arginyl residue (Figure 1A). The msr(W/R) nonapeptide was then incubated with pepsin for 10 min at room temperature. The peptide was not fully degraded, and only two protonated fragments at m/z 1497.7 and 1311.6 (not shown) could be observed in addition to the full-length peptide. Finally, the msr(W/R) nonapeptide was incubated with sonicated CHO K1 cell lysates for 30 min at 37 °C, to expose the peptide to all intracellular enzymes. The full-length peptide was no longer detected, and [M + H]+ peptide fragments at m/z 1125.5, 969.5, and 813.3 accumulated (Figure 2A). This indicates that the trifluoroacetyl-(R,R-diethyl)Gly-Lys(Nbiotin)-(D)Lys-Cys tag is not sensitive to peptidases and can be used as a stable mass reporter tag. msr(W/R) Nonapeptide Uptake and Outflow.26,27 Kinetic analysis (Figure 3A) of the peptide msr(W/R) nonapeptide uptake was done on a wide time scale, from ∼1 min to 24 h at 37 °C. The average quantity determined after 75 min incubation was 40.0 ( 4.6 pmol (n ) 21) in 1 million cells. This intracellular peptide quantity corresponds to 27 ( 3 µM (with 1.5 µL intracellular total volume for 1 million CHO cells). This uptake value remained stable for 4 h and then started to decrease. After 8 h incubation with the peptide, the uptake value was reduced to 30 pmol, which 1934 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

further fell to 8.6 ( 2.2 pmol (n ) 4) after 18 h. This decrease was apparently not due to depletion of the extracellular peptide since similar quantities of intact msr(W/R) nonapeptide in the extracellular medium were measured after 1 or 18 h incubation (Table 1 in Supporting Information). This decrease after 18 h (condition 2) compared to 75 min (condition 1) did not result from saturation processes at the membrane (or aging of the cell culture). Indeed, after addition of 7.5 µM fresh peptide (condition 3) the quantity of peptide in the cells was identical to what was measured after 75 min. In addition, 0.5, 1, or 10 µM of the peptide did not show any cytotoxic effect after 48 h incubation with the cells, as determined with the CCK8 cell counting kit (data not shown). We then examined the possible outflow of the msr(W/R) nonapeptide from the CHO K1 cells after an uptake time of either 1 or 18 h (Figure 3B). MS data showed that the quantity of the full-size peptide and the ion abundances of the degradation fragments (not shown) inside the cells did not change within the 2 h incubation of the cells in the peptide-free medium. The same results were obtained in the presence of trypsin treatment, used to remove the membrane-bound peptide that might have created a physical barrier preventing the cellular peptide from leaking out of the cells. Thus, there is no outflow processes of the full-length peptide and of the degradation fragments within 2 h.

Figure 2. Ion abundance (%; see text) of msr(W/R) nonapeptide fragment ions observed in MALDI-TOF MS spectra after (A) incubation for 30 min at 37 °C of 1 µM msr(W/R) nonapeptide with sonication-treated cell lysates from about 107 CHO K1 cells, (B) uptake experiments for 75 min or 18 h at 37 °C of 7.5 µM msr(W/R) nonapeptide, (C) peptide fragments from the msr(W/R) nonapeptide after uptake (75 min) of the msr(W/R) nonapeptide-PKCi peptide conjugate, and (D) peptide fragments from the PKCi peptide after uptake (75 min) of the msr(W/R) nonapeptidePKCi peptide conjugate.

Intracellular vs Membrane Localization of msr(W/R) Nonapeptide. Depending on the CPP sequence, the amount of surfacebound peptide was previously found to be 5-60 times higher than the internalized peptide, showing the importance of the trypsin treatment to get rid of the peptide stuck in the membrane.26 However, the requirement of trypsin and heparin has been shown for fluorescent peptides.29 Thus, it was worth verifying with our method that the quantified peptide is fully inside the cells and not stuck in or on the external leaflet of the membrane through interactions with lipids and/or disulfide bond with membrane proteins. For that purpose, the effect on peptide quantification of the two water-soluble agents, the phosphine reducing agent tris(2-carboxyethyl)phosphine (TCEP) and the cysteine alkylating agent N-ethylmaleimide (NEM) (Figure 4A, Table 2), was examined. Only NEM is known to cross membranes and enter into cells, while TCEP remains extracellular. The msr(W/R) nonapeptide (7.5 µM) was incubated with cells in the presence of DTT (0.5 mM). After washings with DMEM, TCEP (2 mM, RT) was then added for 3 min to the cells. After new washing with DMEM, NEM (40 mM, 37 °C) was then added to the cells for 3 min. After washing and trypsin treatment, the cells were lysed with 0.3% Triton X100 (15 min at 100 °C). Quantification results have shown that the same ratio for the 1H/2H forms(0.27 ( 0.01) and the 1H(29) Kaplan, I. M.; Wadia, J. S.; Dowdy, S. F. J. Controlled Release 2005, 102, 247-253.

(+NEM)/2H(+NEM) (0.29 ( 0.01) adducts could be determined by mass spectrometry (Table 2). As the deuterated form of the msr(W/R) nonapeptide was added at the lysis step, this result suggests that formation of the NEM adduct on msr(W/R) nonapeptide occurred during the lysis: NEM was not totally washed out during the washing steps and remained sequestered in the membrane. NEM was then released during the lysis and reacted equally with both the intracellular 1H-form and the added extracellular 2H-form of msr(W/R) nonapeptide. Thus, to quench NEM putatively stuck in the membrane, two different concentrations of DTT (0.1 and 0.5 mM) were added at the lysis step. The data demonstrated that, with increasing concentrations of DTT during the cell lysis step, the percentage of the 1H-form that had reacted with NEM, i.e., 1H(+NEM), fell down stepwise to 3% (Table 2). Thus, the quantification method of the intracellular msr(W/R) nonapeptide is fairly accurate with a maximum of 3% of the msr(W/R) nonapeptide being stuck in or on the membrane. Catabolism of msr(W/R) Nonapeptide. We observed in the MS spectra (Figure 1B) that degradation of the intracellular peptide occurred. Results showing the difference in the peptide ion abundance, obtained for the incubation times 75 min and 18 h, are depicted in Figure 2B. Statistical analysis with an alternate Welch t-test of the means of each peptide fragment ion abundance according to time, showed no significant differences (Figure 2B). Thus, there was no accumulation with time of peptide fragments, Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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Figure 3. (A) Kinetic data of the cellular uptake of msr(W/R) nonapeptide. (B) uptake and outflow experiments. After 75 min or 18 h internalization of msr(W/R) nonapeptide (7.5 µM), outflow experiments were run within a 2 h range. (C) Effect of pH neutralization of lysosomes on the celldegradation ion fragment abundances (see text) of the intracellular msr(W/R) nonapeptide. Cells were preincubated for 1 h with 0, 10, or 20 mM NH4Cl, prior to uptake experiments. (D) Effect of increasing KCl concentrations on the uptake of msr(W/R) nonapeptide. (E) Intracellular quantification of msr(W/R) nonapeptide and PKCi after incubation with the msr(W/R) nonapeptide-PKCi conjugate.

the full-length peptide ion being preponderant. For the two incubation times (75 min and 18 h), the mass spectrometry analysis established the presence of all fragments (each not exceeding 10-15% ion abundance) up to the first arginyl residue ([M + H]+ at m/z 969.4), the full-length peptide ion being the most abundant with an ion abundance higher than 50% of the total peptide fragments (Figure 2B). It was rather surprising that the peptide was mostly degraded in the first 75 min of incubation without further degradation after 18 h. To analyze whether these degradation products might be related to the trafficking of the msr(W/R) nonapeptide to lysosomes, we have done parallel experiments in the absence or the presence of NH4Cl (10 or 20 mM) to neutralize the acidic pH of this cell compartment (data not shown).30,31 Cells were preincubated for 1 h with NH4Cl prior to the usual uptake experiment. No significant differences were detected, neither in the degradation fragments observed nor in their relative ion intensities (Figure 3C). These observations do not support the hypothesis of the presence of the peptide in lysosomes. This idea is further supported by experiments with cell lysates in which the peptide is exposed to all cell compartment (30) Ohkuma, S.; Poole, B. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 3327-3331. (31) Zaro, J. L.; Shen, W. C. Exp. Cell Res. 2005, 307, 164- 173. (32) Fischer, R.; Ko ¨hler, K.; Fotin-Mleczek, M.; Brock, R. J. Biol. Chem. 2004, 279, 12625-12635.

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enzymes. With cell lysates, msr(W/R) nonapeptide fragments accumulated, corresponding to the mass reporter tag peptide (ion abundance, 25%) and the mass reporter tag lengthened with the first (ion abundance, 45%) and second (ion abundance, 17%) arginyl residues of the msr(W/R) nonapeptide. This result suggests that the msr(W/R) nonapeptide did not enter through the endosomal pathway or that it has escaped from endosomes before reaching the lysosomes.32 Influence of the Extracellular K+ Concentration on the Cell Entry of msr(W/R) Nonapeptide. Since the membrane potential is one plausible parameter for the mechanism of entry of these positively charged CPP,9,33 the effect of increasing KCl concentration (from 5 to 55 mM) on msr(W/R) nonapeptide cell uptake has been studied. Experiments, done in Hank’s buffered salt solution (HBSS), showed that the msr(W/R) nonapeptide uptake (for 15 min) decreased with increasing extracellular concentrations of KCl (Figure 3D). At 55 mM KCl, the cellular uptake of msr(W/R) nonapeptide was reduced by 55%. These results are in agreement with previous studies9,33 that showed in other cell types that guanidinium-rich peptides uptake can be membrane potential-dependent and thus energy-dependent. Discrepancies between uptake mechanisms reported in the literature (33) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. J. Am. Chem. Soc. 2004, 126, 9506-9507.

Figure 4. (A) Schematic representation of TCEP and NEM effects on membrane-bound CPP. The reductive agent TCEP and cysteine alkylating agent NEM are both water-soluble. However, while TCEP remains extracellular, NEM is able to cross over cell membrane and enter cells where it is quenched by the high concentration of glutathion (GSH). If msr(W/R) nonapeptide is stuck in membrane via noncovalent interaction with lipids or disulfide bridge formation with membrane proteins, TCEP should reduce this latter species while NEM should react with the free cysteinyl residue present in the mass stable reporter tag of msr(W/R) nonapeptide. (B) Schematic representation of msr(W/R) nonapeptide-PKCi conjugate. The (M + H)+ values of the full-length and degradation products of each peptide are also indicated as observed in spectra from MALDI-TOF analyses.

for apparent identical CPP sequences should mainly be related to the chemical reporter (and/or the methodologies) used to quantify the peptide uptake and/or to the cell type. For example

it has been shown for fluorescein-labeled cationic CPPs (penetratin, Tat, and R9) that the endosomal pathway is preferentially used to enter into HeLa cells.32 But, with a cell fractionation protocol Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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Table 2. Effect of Sulfhydryl Reagents on the Ion Abundance of the msr(W/R) Nonapeptide with a NEM Adducta condition usual procedure 1H 2 mM TCEP, 40 mM NEM lysis without DTT 1H 1H(+NEM) 2 mM TCEP, 40 mM NEM lysis with 0.1 mM DTT 1H 1H(+NEM) 2 mM TCEP, 40 mM NEM lysis with 0.5 mM DTT 1H 1H(+NEM)

ion abundance, % 100 47 ( 1 53 ( 2 90 ( 2 10 ( 2 , 97 ( 3 3.0 ( 2.5

a 1H ) Cys-SH and 1H(+NEM) ) Cys-S-NEM in nonapeptide sequence. See text for explanations.

msr(W/R)

and a radioactive R9 analogue, Zaro and Shen have reported that this cell-penetrating peptide mostly entered CHO cells by membrane transduction rather than endocytosis.31 Delivery of a Peptide Cargo into Cells. Finally, we analyzed the potency of the msr(W/R) nonapeptide to convey inside cells a protein kinase C peptide inhibitor (PKCi) previously described.34 The non-conjugated PKCi was inefficient to enter CHO cells as already determined by our MALDI-TOF based quantification method.28 The PKCi peptide contains five basic residues. For uptake quantification, distinction between extracellular PKCi peptide and truly internalized peptide is donesas for the CPP quantificationsby applying a trypsin treatment. The PKCi and msr(W/R) nonapeptide were linked via a disulfide bond that should immediately be reduced once the conjugate (Figure 4B) enters into the cells cytoplasm.28 Uptake experiments after 75 min incubation with the conjugate led to quantify 53 ( 4 pmol of msr(W/R) nonapeptide and 17 ( 3 pmol of PKCi in 1 million cells (Figure 3E). The degradation fragments of the intracellular msr(W/R) nonapeptide in the conjugate were identical to those observed with the msr(W/R) nonapeptide alone (Figure 2C). For the intracellular PKCi, degradation products were also observed sequentially from the C-terminus up to the first arginyl residue (Figure 2D). Thus, uptake quantification of both the CPP and the cargo from the peptide conjugate in cells showed differences in the apparent uptake values for the two peptides, being about 3-fold more for the CPP compared to the PKCi peptide. The PKCi peptide is linked via a disulfide bridge to the mass stable reporter tag of msr(W/R) nonapeptide and this covalent bond is stable in the extracellular medium. Thus, the apparent lower value of the intracellular quantity of cargo likely results from its higher degradation sensitiveness inside or outside cells compared to the CPP. Indeed, we could not detect putative PKCi peptide fragments with m/z under 500, since the mass stable reporter tag was present (34) Theodore, L.; Derossi, D.; Chassaing, G.; Llirbat, B.; Kubes, M.; Jordan, P.; Chneiweiss, H.; Godement, P.; Prochiantz, A. J. Neurosci. 1995, 15, 71587167. (35) Ong, S.-E.; Mann, M. Nat. Chem. Biol. 2005, 1, 252-262. (36) Balayssac, S.; Burlina, F.; Convert, O.; Bolbach, G.; Chassaing, G.; Lequin, O. Biochemistry 2006, 45, 1408-1420. (37) Ha¨llbrink, M.; Oehlke, J.; Papsdorf, G.; Bienert, M. Biochim. Biophys. Acta 2004, 1667, 222-228.

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only on the CPP. Nevertheless the quantity of the intracellular msr(W/R) nonapeptide was identical in the conjugate compared to the peptide alone. Moreover the CPP fragments observed after conjugate internalization were the same as those found when the CPP was internalized alone. These results suggest that the intracellular addressing of the CPP was identical in the absence or the presence of the PKCi cargo.23 CONCLUSION The mass stable reporter (msr) tag described herein, trifluoroacetyl-(R,R-diethyl)Gly-Lys(Nbiotin)-(D)Lys-Cys, might be incorporated in all known CPP sequences to achieve quantification and to detect all possible peptide degradation fragments by MALDI-TOF mass spectrometry analysis. This mass reporter tag represents an important tool that allows an accurate tracking of the full-length CPP and of its degradation products inside and outside cells. Absolute quantification of CPP degradation products may be achieved, using the deuterium-containing forms of each peptide fragment as for the full-length CPP.26-28,35 This study showed that msr(W/R) nonapeptide is the most efficient CPP so far determined by this method of uptake quantification.26-28,36 Indeed, the uptake value measured after 1 h (corresponding to both uptake and degradation processes) of the full-length peptide is advantageously compared with different CPP we have so far studied, that is, msr(W/R) nonapeptide ≈ the third helix of Knotted homeodomain (25 µM) > R9 (4.5 µM) ≈ penetratin (3.5 µM) > Tat48-59 (0.7 µM), although these last four CPP had not the msr tag described here but a biotin-(Gly)4 spacer arm.26-28,36 The kinetic of entry of msr(W/R) nonapeptide is rapid, reaching a steady-state after 30-60 min of incubation. This plateau was stable for 4 h and decreased slowly afterward. It was also observed that there was no outflow processes of the full-length peptide and of the degradation fragments within 2 h. However, to explain such decrease in the intracellular quantity with time, longer outflow experiments have to be done. In addition, the partition coefficient or the CPP-to-cell ratio37 have to be fully studied to analyze the influence of such parameters on the intracellular delivery of these peptides. Finally, the trafficking of CPP in cells is an important question, which can be addressed by mass spectrometry, since these peptides can be used as therapeutic tools to convey biologically active compounds into cells. ACKNOWLEDGMENT We thank the French Ministe`re de l’EÄ ducation Nationale, de la Recherche et de la Technologie (MENRT Ph.D. grants for D.D., B.A., and S.A.), Centre National de la Recherche Scientifique (CNRS), and Re´gion Ile-de-France for financial support. SUPPORTING INFORMATION AVAILABLE Table 1, showing differences in uptake with experimental parameters and describing the chemical synthesis of the trifuloroacetyl-(R,R-diethyl)glycine group and the protocol of cell uptake used in the study. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 19, 2006. Accepted December 6, 2006. AC061108L