morphogenesis [3]. Mapping of the domains responsible for this
cell-penetrating ability led to the identification of two protein
transduction domains, (also known as cell-penetrating peptides or
CPPs); Tat corresponding to the 11 amino acid basic domain of
HIV-1 Tat protein, and Penetratin corresponding to the 16 amino
acid third helix of the Antennapedia domain. Subsequently, it was
demonstrated that Tat fused to β-galactosidase and injected intraperitoneally in mice was internalized into multiple cell types including liver, heart, lung, kidney, and brain, delivering β-galactosidase
in a functional form, highlighting the potential of Tat as a vector
[4]. Currently, multiple cargoes in the form of peptides, proteins,
nucleic acid, nanoparticles, and radioisotopes have been delivered
using various CPPs [5, 6].
The ability of cationic or hydrophobic CPPs to transduce a
wide variety of tissue types in vivo limits their utility because of
lack of cell specificity. Phage display using libraries of various
lengths and different bacteriophage strains have been utilized successfully to identify tissue-specific CPPs [7]. In our prior work, we
identified a mildly basic, non-naturally occurring peptide (NH 2 -A
PWHLSSQYSRT-COOH) capable of specifically targeting normal
cardiomyocytes (CMCs) in vivo in mice, which we hence termed
Cardiac Targeting Peptide or CTP [7, 8]. Our detailed bio-distribution studies show that peak uptake occurs at 15 min with complete disappearance of fluorescently labeled CTP by 6 hrs [9].
Cellular responses to ligands such as peptides, proteins, pharmaceutical drugs, or entire pathogens are generally mediated
through interactions with specific proteins expressed on the cell
surface. The ligand-based receptor capture (LRC)-TriCEPS methodology has been designed to directly identify such ligand–receptor
interactions under near-physiological conditions on living cells.
The key component of the LRC methodology is a trifunctional
cross-linker that combines the following moieties; a
N-hydroxysuccinimide (NHS), a hydrazone, and a biotin or an
azide [10, 11].
In a typical LRC-TriCEPS experiment, at least two treatment
arms are performed in parallel: one with the ligand of interest and a
second with a control ligand with a known target. The first step of
the LRC-TriCEPS experiment includes the conjugation of the
ligand to TriCEPS using the NHS-ester. The TriCEPS-ligand conjugates are then incubated with previously oxidized cells. During
this phase, the hydrazone captures covalently the surface glycoproteins via the aldehydes that are introduced at the carbohydrates by
mild oxidation (receptor capture). After the receptor-capture reaction, the cells are lysed and the azide or the biotin groups are used
to purify the cross-linked proteins for downstream mass spectrometry (MS)-based analysis (cell lysis, protein enrichment, and digestion). Upon identification, the cell surface proteins in the ligand
samples are compared to those in the control sample using
98
Kyle S. Feldman et al.
cell-penetrating ability led to the identification of two protein
transduction domains, (also known as cell-penetrating peptides or
CPPs); Tat corresponding to the 11 amino acid basic domain of
HIV-1 Tat protein, and Penetratin corresponding to the 16 amino
acid third helix of the Antennapedia domain. Subsequently, it was
demonstrated that Tat fused to β-galactosidase and injected intraperitoneally in mice was internalized into multiple cell types including liver, heart, lung, kidney, and brain, delivering β-galactosidase
in a functional form, highlighting the potential of Tat as a vector
[4]. Currently, multiple cargoes in the form of peptides, proteins,
nucleic acid, nanoparticles, and radioisotopes have been delivered
using various CPPs [5, 6].
The ability of cationic or hydrophobic CPPs to transduce a
wide variety of tissue types in vivo limits their utility because of
lack of cell specificity. Phage display using libraries of various
lengths and different bacteriophage strains have been utilized successfully to identify tissue-specific CPPs [7]. In our prior work, we
identified a mildly basic, non-naturally occurring peptide (NH 2 -A
PWHLSSQYSRT-COOH) capable of specifically targeting normal
cardiomyocytes (CMCs) in vivo in mice, which we hence termed
Cardiac Targeting Peptide or CTP [7, 8]. Our detailed bio-distribution studies show that peak uptake occurs at 15 min with complete disappearance of fluorescently labeled CTP by 6 hrs [9].
Cellular responses to ligands such as peptides, proteins, pharmaceutical drugs, or entire pathogens are generally mediated
through interactions with specific proteins expressed on the cell
surface. The ligand-based receptor capture (LRC)-TriCEPS methodology has been designed to directly identify such ligand–receptor
interactions under near-physiological conditions on living cells.
The key component of the LRC methodology is a trifunctional
cross-linker that combines the following moieties; a
N-hydroxysuccinimide (NHS), a hydrazone, and a biotin or an
azide [10, 11].
In a typical LRC-TriCEPS experiment, at least two treatment
arms are performed in parallel: one with the ligand of interest and a
second with a control ligand with a known target. The first step of
the LRC-TriCEPS experiment includes the conjugation of the
ligand to TriCEPS using the NHS-ester. The TriCEPS-ligand conjugates are then incubated with previously oxidized cells. During
this phase, the hydrazone captures covalently the surface glycoproteins via the aldehydes that are introduced at the carbohydrates by
mild oxidation (receptor capture). After the receptor-capture reaction, the cells are lysed and the azide or the biotin groups are used
to purify the cross-linked proteins for downstream mass spectrometry (MS)-based analysis (cell lysis, protein enrichment, and digestion). Upon identification, the cell surface proteins in the ligand
samples are compared to those in the control sample using
98
Kyle S. Feldman et al.