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Quantification of Cell-Penetrating Peptides and Their Cargoes 269
N-terminal α-amino group was used for attachment. The peptide was synthesized
by standard t-Boc solid phase peptide synthesis. For FITC labeling of the same
peptide, the α-amino group of the peptide was deprotected and 100 mg of peptidylresin
was incubated with five-fold excess of 4(5)-carboxyfluorescein succinimidyl
ester (Molecular Probes, Holland) for 24 h at room temperature in a 1:1 mixture of
dimethyl formamide (DMF)/dimethyl sulfoxide (DMSO), with five-fold excess of
DIEA. For biotin or Abz, three-fold excess of HOBt and TBTU activated biotin or
t-Boc-Abz in DMF was incubated with the peptidyl-resin for 30 min at RT. The
peptides were cleaved from the resins (with liquid HF at 0°C for 30 min) in the
presence of scavenger (p-cresol). The purity of the peptides was checked by HPLC
analysis on Nucleosil 120-3 C 18 column (0.4 × 10 cm). Molecular mass of each
synthetic peptide was determined on a Voyager MALDI-TOF. 5,17
Cellular internalization assay: The cells used for internalization experiments
were seeded at the density of 10,000 cells/glass coverslip (10 mm diameter) in
24-well plates. One day post seeding, the cells were semiconfluent and media were
changed to serum-free media containing 10 µM of labeled peptide. After 60 min of
incubation at either 37 or 4°C, the cells were washed three times with phosphatebuffered
saline (PBS) and fixed with 3% (v/v) paraformaldehyde solution in PBS
for 15 min. For indirect detection of biotinylated peptide, the fixed cells were permeabilised
with 0.5% Triton X-100 in PBS and sites for nonspecific binding were
blocked with 5% bovine serum albumin (BSA) in PBS. The biotin moieties were
visualized by incubation with Texas red-labeled streptavidin (1:200 in blocking
solution, Molecular Probes, Holland) for 1 h at room temperature. The cell nuclei
were stained with Hoechst 33258 (0.5 µg/ml, Molecular Probes, Holland). After
washing with PBS, the preparates were mounted in Fluoromount G water base
(Electron Microscopy Science, PA, USA).
Quantitative uptake of fluorescence-labeled peptide: Cells were seeded in
12-well plates at the density of 100,000 cells/well and incubated overnight to adhere.
Twelve hours after seeding, the cells were washed once with serum-free media and
treated with 10 µM peptide, dissolved in serum-free media, for 60 min at 37°C. The
cells were washed twice with HEPES-buffered Krebs–Ringer solution (HKR) and
detached by trypsin treatment for 3 min. The cells were centrifuged at 4000 × g for
10 min at 4°C and resuspended in 200 µl of HKR. The fluorescence was measured
in a fluorescence spectrophotometer (F-2000, Hitachi, Japan). The concentration of
fluorescence-labeled peptide was calculated from a standard curve of the peptide in
HKR. Alternatively, the cells were detached before peptide treatment and the peptide
was added to the cells in suspension and incubated on a shaking 37°C water bath.
Then the outer membrane-bound peptide was degraded by a 4-min trypsin treatment,
after which the cells were spun down, washed, and resuspended in HKR and the
fluorescence intensity measured. For the wavelength applied, see Figure 12.1.
12.2.1.4 HPLC Detection
Oehlke et al. have used high-performance liquid chromatography to quantify uptake
of fluorescein-labeled cell-penetrating peptides 4 (for further details, see Chapter 4).
In order to separate cell surface-bound and internalized peptide, treated cells were