sonication step is integrated with the rest of the procedures, and acoustic energy is
used to facilitate the mixing and washing of beads. In this way the experiment can be
completed in only 40 min. Cross-linked cells are placed on the microfluidic chip to
generate sonication of DNA through a magnetic field. The use of crescent structures
increases the gas-liquid contact surface, increasing the power of the magnetic field.
Low acoustic waves are then used to allow greater interaction between the beads and
cell extract. The immunoprecipitation step is completed after 30 min. The beads are
simply washed with a buffer to remove impurities and then collected for analysis. The
obvious advantages of this technique include small amounts of starting material, ease
of experimental procedure, very short experiment times and high resolution.
1.2.2 Cellular Thermal Shift Assay (CETSA)
CETSA allows for the evaluation of protein interactions through macromolecular
stabilization or destabilization and therefore variation in the melting temperature
(Tm) [24]. Specifically, this technique is based on the biophysical principle that if a
protein/enzyme is subjected to temperature increase, it will undergo protein
unfolding with different melting curves. CETSA has several advantages: (1) it is
not necessary to modify the molecule for immobilization on beads (as in chemoproteomic approach, AlphaLISA, MST); (2) it is not an expensive technique; and
(3) it is very simple to perform. The CETSA protocol includes the following steps:
incubation of cells or cell extracts (to ensure permeability of the compound) with the
molecule of interest; incubation of cell pellets at different temperatures, usually
between 37
C and 70
C (the choice of temperature depends on the protein
under investigation); total protein extraction; and detection of stabilization by
Western blot.
Over the years, this technique has undergone various modifications, such as the
use of different compound concentrations to evaluate dose-dependent enzymatic
stability or to study not only the interaction of the molecule with a single protein but
with all the cellular proteasomes. A two-dimensional thermal proteomic profile
strategy was developed to investigate all the off-target effects of panobinostat
(an epigenetic drug used for multiple myeloma) [25]. After treatment of the cells
with panobinostat at five different concentrations and incubation at 12 different
temperatures, all proteins undergoing a change in Tm, and therefore all proteins
that bind the drug, were analysed by liquid chromatography-mass spectrometry
(LC-MS)/MS. Using this assay, four new different proteins which interact with the
compound were identified. One of the limitations in using CETSA is that it is
unsuitable for HTS of protein interactions due to the time and quantity of materials
required. In 2017, Holbert and colleagues [26] developed a new method named highthroughput dose-response (HTDR)-CETSA, which allows for HTS using a 384-well
plate format of drug-protein interactions by chemiluminescence signal (Fig. 3). In
this assay the target protein, SMYD3, is fused and overexpressed in the cell using
the BACMam system (ThermoFisher Scientific), with a small enzyme donor fragment of β-galactosidase (β-gal) called enhanced ProLabel
® (ePL; DiscoverX).
532
C. Dell’Aversana et al.
used to facilitate the mixing and washing of beads. In this way the experiment can be
completed in only 40 min. Cross-linked cells are placed on the microfluidic chip to
generate sonication of DNA through a magnetic field. The use of crescent structures
increases the gas-liquid contact surface, increasing the power of the magnetic field.
Low acoustic waves are then used to allow greater interaction between the beads and
cell extract. The immunoprecipitation step is completed after 30 min. The beads are
simply washed with a buffer to remove impurities and then collected for analysis. The
obvious advantages of this technique include small amounts of starting material, ease
of experimental procedure, very short experiment times and high resolution.
1.2.2 Cellular Thermal Shift Assay (CETSA)
CETSA allows for the evaluation of protein interactions through macromolecular
stabilization or destabilization and therefore variation in the melting temperature
(Tm) [24]. Specifically, this technique is based on the biophysical principle that if a
protein/enzyme is subjected to temperature increase, it will undergo protein
unfolding with different melting curves. CETSA has several advantages: (1) it is
not necessary to modify the molecule for immobilization on beads (as in chemoproteomic approach, AlphaLISA, MST); (2) it is not an expensive technique; and
(3) it is very simple to perform. The CETSA protocol includes the following steps:
incubation of cells or cell extracts (to ensure permeability of the compound) with the
molecule of interest; incubation of cell pellets at different temperatures, usually
between 37
C and 70
C (the choice of temperature depends on the protein
under investigation); total protein extraction; and detection of stabilization by
Western blot.
Over the years, this technique has undergone various modifications, such as the
use of different compound concentrations to evaluate dose-dependent enzymatic
stability or to study not only the interaction of the molecule with a single protein but
with all the cellular proteasomes. A two-dimensional thermal proteomic profile
strategy was developed to investigate all the off-target effects of panobinostat
(an epigenetic drug used for multiple myeloma) [25]. After treatment of the cells
with panobinostat at five different concentrations and incubation at 12 different
temperatures, all proteins undergoing a change in Tm, and therefore all proteins
that bind the drug, were analysed by liquid chromatography-mass spectrometry
(LC-MS)/MS. Using this assay, four new different proteins which interact with the
compound were identified. One of the limitations in using CETSA is that it is
unsuitable for HTS of protein interactions due to the time and quantity of materials
required. In 2017, Holbert and colleagues [26] developed a new method named highthroughput dose-response (HTDR)-CETSA, which allows for HTS using a 384-well
plate format of drug-protein interactions by chemiluminescence signal (Fig. 3). In
this assay the target protein, SMYD3, is fused and overexpressed in the cell using
the BACMam system (ThermoFisher Scientific), with a small enzyme donor fragment of β-galactosidase (β-gal) called enhanced ProLabel
® (ePL; DiscoverX).
532
C. Dell’Aversana et al.