(D-133) Development of a lipid-conjugated substrate to measure the activity of protein kinase B in single cells by capillary electrophoresis

Introduction:: Protein kinase B (PKB), also known as Akt, phosphorylates proteins within signaling pathways, which regulate cell survival, apoptosis, cell cycle progression, and cell migration. Since activation of PKB is reported in many tumor types, new strategies to measure PKB activity in single cells in addition to mRNA or protein measurements would be of high value in understanding cancer-cell signaling. PKB activity measurements in single cells have been performed previously by capillary electrophoresis (CE)-based assays. A peptidase-resistant PKB substrate, fluorescein-GRP(N-Me-Arg)AFTF(N-Me-Ala) (48F, Figure1A) was introduced into cells and then phosphorylated by active cellular PKB to form phosphorylated-48F (p48F, Figure1B). Loading a cell's contents into a capillary followed by electrophoresis enabled separation and detection of 48F, p48F, and any fluorescent fragments formed by peptidases within the cell. The p48F (% formed) acted as a measure of PKB activity. A challenge has been the inefficient loading of the positively charged 48F into cells since 48F must be introduced into cells by microinjection or pinocytic loading. A second barrier has been the consistent separation of 48F, p48F, and the fluorescent fragments in a simple electrophoretic buffer system. In this work, a C14 lipid-modified version of 48F (48F-C14, Figure1C) was developed to increase the membrane permeability of 48F for facile loading into cells. Additionally, the placement of the C14 group on the opposite end of the peptide from the fluorescein enabled the enhanced separation of 48F-C14 and p48F-C14 (Figure1D) from the fluorescent fragments formed in the cell since these fragments do not contain the C14 group.

Materials and Methods:: The synthesis of peptide substrate (fluorescein-GRP(N-Me-Arg)AFTF(N-Me-Ala)K(C14)-CONH2) followed standard Fmoc peptide synthetic protocols with initial amino acid coupling via a C-terminal Fmoc-Lys(Mtt)-OH to Rink amide PEGA resin. The Lys was selectively deprotected and subsequently coupled with myristic acid. The N-terminus of peptides was labeled with 6-carboxyfluorescein. Peptide was simultaneously deprotected and cleaved from resin by treatment with 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water, purified by reverse-phase chromatography on a C18 column, and confirmed by liquid chromatography-mass spectrometry.
HeLa Cells were plated on a #1 glass coverslip and incubated overnight. After washing, cells were incubated with peptides (10 μM). The solution was aspirated, and the cells were washed to remove residual peptides. The cells were imaged by confocal fluorescence microscopy to verify peptide loading into the cells.
Fragments of the peptide (10 μM) were formed by incubation with Pronase E (100 μg/mL, 1 h). The reaction was stopped by incubation at 95 °C (5 min). Fragment formation was assessed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).
Electrophoretic separation of peptides was performed on a commercial CE system in a fused silica capillary (30 cm long, 30 μm inner diameter and 360 μm outer diameter). The peptides were detected by excitation at 488 nm and emission at 520 nm. Electrophoretic traces were analyzed using customized MATLAB software. Various buffer systems were assessed for their ability to separate the peptides; these included buffers with organic solvents (1-propanol), salts (monosodium phosphate and Tris), and detergents (Tween20).

Results, Conclusions, and Discussions:: 48F-C14 was readily visualized by fluorescence, which was significantly greater than that of 48F when loaded into HeLa cells (Figure2A-B). This data suggests that C14 promotes the movement of 48F-C14 into the cells.
The peptidase resistance of 48F-C14 was assessed by Pronase E reaction followed by MALDI-TOF MS analysis. The detected most abundant peptides were 48F-C14 and fluorescein-GRP(N-Me-Arg)A, which was the dominant fragment from the parent 48F with Pronase E reaction. This outcome indicates that 48F-C14 might be reduce the sensitivity for the peptidases found within cells.
The electrophoretic buffer used for the separation of 48F-C14, p48F-C14, and the fluorescent fragments was optimized. After assessing various buffer systems with differing attributes, the best buffer that offer the best solubility for the C14-labelled peptides was 10 mM NaH2PO4 with 80 mM Tween20 (pH 7.5) with a resolution between 48F-C14 and p48F-C14 at 8.2 ± 0.1 and theoretical plate numbers at 23000 ± 100 for 48F-C14 and at 15 ± 200 for p48F-C14 (100 nM, n = 4, Figure2C).
48F-C14 demonstrated excellent cell permeability while maintaining the protease resistance of the parent 48F peptide. The C14 moiety also facilitated the separation of fluorescent fragments from intact 48F-C14 and p48F-C14, offering the possibility to provide a more reliable quantitative measurement of p48F-C14 formed in a cell under "real-world" experimental conditions. Future experiments will utilize 48F-C14 for PKB activity assay in single cells from normal and diseased patients and in the absence and presence of inhibitors and other perturbants.

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