label-free quantification (Fig. 1). Randomly identified cell surface
proteins are expected to have equal abundance in both samples,
whereas the corresponding targets are found enriched in the ligand
sample [10, 11].
The LRC-TriCEPS methodology offers several unique advantages. It can be applied in a multitude of ligands ranging from small
molecules to peptides, proteins, antibodies, and even whole viruses.
Moreover, it does not require any genetic manipulation and therefore can be applied to a multitude of cell lines, including primary
cells. Furthermore, targets are located within the context of the
natural cell-specific surface microenvironment, so they are fully
functional and exhibit their characteristic binding properties.
Finally, it is hypothesis-free, meaning that no previous knowledge
about the target is required.
A drawback of the methodology can be missing an interaction
in case the target receptor is not expressed in the selected model
system or TriCEPS coupling leads to hindering of the ligand–
receptor interaction, which would interfere with ligand identification. In these scenarios, TriCEPS would not be useful. Additionally,
the LRC-TriCEPS methodology enables the identification of only
glycosylated targets; therefore, a small percentage of binding partners, less than 10%, cannot be identified.
In this chapter, we present details on the various methodologies
that can be used to confirm tissue-specific internalization of a cellspecific CPP using CTP targeting the heart as an example. We also
present details on LRC methodology.
2 Materials
2.1 Validation of
Tissue-Specific CPPs
This section provides details on how to validate a candidate CPP for
cell-specific transduction and contains protocols that can be modified for use in a variety of cell lines and organs, using fluorescently
labeled peptides. We also provide a protocol for fluorophore conjugation of biotinylated CPPs. The methods detailed below use
CTP in H9C2 cells, a rat cardiomyoblast cell line and wild-type
mouse animal models as an example.
2.1.1 Transduction Assay
Utilizing FluorescenceActivated Cell Sorting or
Confocal Imaging
1. Dulbecco’s Modified Eagle’s Medium—high glucose
(DMEM).
2. Dulbecco’s Phosphate Buffered Saline (PBS).
3. Heat-inactivated fetal bovine serum (FBS).
4. Antibiotic-Antimycotic.
5. Rat Cardiomyoblast Cell Line (H9C2; ATCC).
6. Rat Cardiomyoblast Cell Line (H9C2 Cells) Media: Add
50 mL of heat-inactivated FBS and 5 mL of antibioticantimycotic to 500 ml of DMEM. Filter the mixture and
store at 4
C.
Validating Cardiac Targeting Peptide
99
proteins are expected to have equal abundance in both samples,
whereas the corresponding targets are found enriched in the ligand
sample [10, 11].
The LRC-TriCEPS methodology offers several unique advantages. It can be applied in a multitude of ligands ranging from small
molecules to peptides, proteins, antibodies, and even whole viruses.
Moreover, it does not require any genetic manipulation and therefore can be applied to a multitude of cell lines, including primary
cells. Furthermore, targets are located within the context of the
natural cell-specific surface microenvironment, so they are fully
functional and exhibit their characteristic binding properties.
Finally, it is hypothesis-free, meaning that no previous knowledge
about the target is required.
A drawback of the methodology can be missing an interaction
in case the target receptor is not expressed in the selected model
system or TriCEPS coupling leads to hindering of the ligand–
receptor interaction, which would interfere with ligand identification. In these scenarios, TriCEPS would not be useful. Additionally,
the LRC-TriCEPS methodology enables the identification of only
glycosylated targets; therefore, a small percentage of binding partners, less than 10%, cannot be identified.
In this chapter, we present details on the various methodologies
that can be used to confirm tissue-specific internalization of a cellspecific CPP using CTP targeting the heart as an example. We also
present details on LRC methodology.
2 Materials
2.1 Validation of
Tissue-Specific CPPs
This section provides details on how to validate a candidate CPP for
cell-specific transduction and contains protocols that can be modified for use in a variety of cell lines and organs, using fluorescently
labeled peptides. We also provide a protocol for fluorophore conjugation of biotinylated CPPs. The methods detailed below use
CTP in H9C2 cells, a rat cardiomyoblast cell line and wild-type
mouse animal models as an example.
2.1.1 Transduction Assay
Utilizing FluorescenceActivated Cell Sorting or
Confocal Imaging
1. Dulbecco’s Modified Eagle’s Medium—high glucose
(DMEM).
2. Dulbecco’s Phosphate Buffered Saline (PBS).
3. Heat-inactivated fetal bovine serum (FBS).
4. Antibiotic-Antimycotic.
5. Rat Cardiomyoblast Cell Line (H9C2; ATCC).
6. Rat Cardiomyoblast Cell Line (H9C2 Cells) Media: Add
50 mL of heat-inactivated FBS and 5 mL of antibioticantimycotic to 500 ml of DMEM. Filter the mixture and
store at 4
C.
Validating Cardiac Targeting Peptide
99