Tracking a new cell-penetrating (W/R) nonapeptide, through an enzyme-stable mass spectrometry reporter tag

Article abstract of DOI:10.1021/ac061108l

We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(α,α-diemyl)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-NH ₂) 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.

Full text of DOI:10.1021/ac061108l

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
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 endo-
somal 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, incorpora-
tion 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, reso-
nance energy transfer, HPLC detection, immunodetection, fluo-
rescence correlation microscopy, laser micropipette system, or
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 quantita-
tive 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 con-
centration (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 lyso-
somes.
(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, 9185-
9194.
(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.;
Cell-penetrating peptidessCPP (also named protein transduc-
tion 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
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.
* To whom correspondence should be addressed. E-mail: sagan@ccr.jussieu.fr
(S.S.); dlaroche@ccr.jussieu.fr (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.; B¨ahr, M. Mol. Cell. Neurosci. 2004, 27, 85-131.
(5) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Trends Pharmacol.
Sci. 2000, 21, 99-103.
(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.
(6) Derossi, D.; Chassaing, G.; Prochiantz, A. Trends Cell Biol. 1998, 8,
84-87.
1932 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007
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, trifluoro-
acetyl-(R,R-diethyl)Gly-Lys(Nꢀbiotin)-(D)Lys-Cys, has been incor-
porated 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 MALDI-
TOF 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.
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.
EXPERIMENTAL SECTION
Cell Culture. Chinese hamster ovary (CHO) K1 cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented 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
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-cyano-
4-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).
(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.
RESULTS AND DISCUSSION
Stability of the Mass Reporter Tag. The trifluoroacetyl-(R,R-
diethyl)Gly-Lys(Nꢀbiotin)-(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 1933

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).
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.
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(Nꢀbiotin)-(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

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) nonapeptide-
PKCi peptide conjugate.
Intracellular vs Membrane Localization of msr(W/R) Nona-
peptide. Depending on the CPP sequence, the amount of surface-
bound 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 exam-
ined. Only NEM is known to cross membranes and enter into
cells, while TCEP remains extracellular. The msr(W/R) nonapep-
tide (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-
(+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) non-
apeptide 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
1
equally with both the intracellular H-form and the added extra-
cellular 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
1
with NEM, i.e., H(+NEM), fell down stepwise to 3% (Table 2).
Thus, the quantification method of the intracellular msr(W/R)
msr
nonapeptide is fairly accurate with a maximum of 3% of the
(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,
(29) Kaplan, I. M.; Wadia, J. S.; Dowdy, S. F. J. Controlled Release 2005, 102,
247-253.
Analytical Chemistry, Vol. 79, No. 5, March 1, 2007 1935

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 cell-
degradation 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
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 endo-
somal 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. Dis-
crepancies between uptake mechanisms reported in the literature
(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.
(33) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A.
J. Am. Chem. Soc. 2004, 126, 9506-9507.
1936 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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 (penetra-
tin, 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 1937

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
Table 2. Effect of Sulfhydryl Reagents on the Ion
Abundance of the msr(W/R) Nonapeptide with a NEM
Adducta
ion abundance,
condition
%
usual procedure
1H
100
2 mM TCEP, 40 mM NEM lysis without DTT
1H
47 ( 1
53 ( 2
CONCLUSION
1H(+NEM)
The mass stable reporter (msr) tag described herein, trifluo-
2 mM TCEP, 40 mM NEM lysis with 0.1 mM DTT
1H
90 ( 2
10 ( 2
,
97 ( 3
3.0 ( 2.5
roacetyl-(R,R-diethyl)Gly-Lys(Nꢀbiotin)-(D)Lys-Cys, might be in-
corporated 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
1H(+NEM)
2 mM TCEP, 40 mM NEM lysis with 0.5 mM DTT
1H
1H(+NEM)
a 1H
) )
Cys-SH and 1H(+NEM) Cys-S-NEM in msr(W/R)
nonapeptide sequence. See text for explanations.
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.
and a radioactive R9 analogue, Zaro and Shen have reported that
this cell-penetrating peptide mostly entered CHO cells by mem-
brane 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
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 trifu-
loroacetyl-(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.
(34) Theodore, L.; Derossi, D.; Chassaing, G.; Llirbat, B.; Kubes, M.; Jordan, P.;
Chneiweiss, H.; Godement, P.; Prochiantz, A. J. Neurosci. 1995, 15, 7158-
7167.
(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.
Received for review June 19, 2006. Accepted December 6,
2006.
(37) H¨allbrink, M.; Oehlke, J.; Papsdorf, G.; Bienert, M. Biochim. Biophys. Acta
2004, 1667, 222-228.
AC061108L
1938 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007