sensitivity, specificity and reproducibility of epigenetic technologies will not only
reveal underlying deregulated epigenetic pathways associated with disease progression and treatment response but will also provide a robust measure of novel biomarkers for risk stratification in terms of disease development and for individualized
precision medicine.
1 Epi-proteomic Analyses
Post-translational histone modifications make up a significant portion of epigenetic
mechanisms involved in several biological processes. Chromatin remodelling is
known to affect up- and downregulation of gene expression, and tumorigenic
diseases can be driven by alterations of epigenetic modification patterns. New
technologies have therefore been designed to identify histone modification enrichment in specific cellular contexts, and new drugs able to restore the physiological
epi-state have been developed. This section of the chapter focuses on the techniques
most commonly used to rapidly identify macromolecular complexes and to discover
drugs that bind and modify protein activity. The assays have been divided into two
main groups: (1) in vitro assays following protein expression and purification and
(2) techniques that allow experiments to be performed directly in cells.
1.1 Biochemical Studies: In Vitro Assay
The choice of technique mainly depends on the starting point and objectives. The
advantage of biochemical experiments is that the protein of interest can be isolated,
purified and overexpressed. Data analysis is straightforward and fast. Indeed, this
approach is commonly used to detect protein-protein or protein-drug interactions in
high-throughput screening (HTS) strategies. Cell-based assays allow investigation
of the target protein, and all the macromolecular complexes of which it is a part,
under physiological conditions. In this case it is important to take into account
several factors that can complicate studies in cells, such as drug permeability and
half-life, cellular enzymatic degradation, small amounts of starting material and long
lead times of experiments. The analysis of chemiluminescent protein interactions
(AlphaScreen/AlphaLISA), variation in diffraction radius (surface plasmon resonance), or changes in molecular solvation (isothermal titration calorimetry) are the
most common techniques used for in vitro macromolecule interaction studies.
1.1.1 AlphaScreen/AlphaLISA
Biomolecular interactions, post-translational modifications or activity of substrates
as competitor compounds can be investigated using AlphaScreen and AlphaLISA
526
C. Dell’Aversana et al.
reveal underlying deregulated epigenetic pathways associated with disease progression and treatment response but will also provide a robust measure of novel biomarkers for risk stratification in terms of disease development and for individualized
precision medicine.
1 Epi-proteomic Analyses
Post-translational histone modifications make up a significant portion of epigenetic
mechanisms involved in several biological processes. Chromatin remodelling is
known to affect up- and downregulation of gene expression, and tumorigenic
diseases can be driven by alterations of epigenetic modification patterns. New
technologies have therefore been designed to identify histone modification enrichment in specific cellular contexts, and new drugs able to restore the physiological
epi-state have been developed. This section of the chapter focuses on the techniques
most commonly used to rapidly identify macromolecular complexes and to discover
drugs that bind and modify protein activity. The assays have been divided into two
main groups: (1) in vitro assays following protein expression and purification and
(2) techniques that allow experiments to be performed directly in cells.
1.1 Biochemical Studies: In Vitro Assay
The choice of technique mainly depends on the starting point and objectives. The
advantage of biochemical experiments is that the protein of interest can be isolated,
purified and overexpressed. Data analysis is straightforward and fast. Indeed, this
approach is commonly used to detect protein-protein or protein-drug interactions in
high-throughput screening (HTS) strategies. Cell-based assays allow investigation
of the target protein, and all the macromolecular complexes of which it is a part,
under physiological conditions. In this case it is important to take into account
several factors that can complicate studies in cells, such as drug permeability and
half-life, cellular enzymatic degradation, small amounts of starting material and long
lead times of experiments. The analysis of chemiluminescent protein interactions
(AlphaScreen/AlphaLISA), variation in diffraction radius (surface plasmon resonance), or changes in molecular solvation (isothermal titration calorimetry) are the
most common techniques used for in vitro macromolecule interaction studies.
1.1.1 AlphaScreen/AlphaLISA
Biomolecular interactions, post-translational modifications or activity of substrates
as competitor compounds can be investigated using AlphaScreen and AlphaLISA
526
C. Dell’Aversana et al.