26. LC solvent A (0.4% formic acid, 2% acetonitrile, 97.6% water).
27. LC solvent B (0.4% formic acid, 95% acetonitrile, 4.6% water).
28. Electrospray ionization (ESI) mass spectrometer (any
ESI-equipped MS with reasonable resolution is usable; in our
case, we used an electrospray ionization—Fourier transform
ion cyclotron resonance 15T solariX XR, Bruker Daltonics).
29. Software: DataAnalysis 4.1 (Bruker Daltonics), ProteinScape 4
(Bruker Daltonics), DeutEx (http://peterslab.org/downloads.
php under HDX tools section), mMass (http://mmass.org),
PyMol (https://pymol.org/; v 2.1.0), MSTools (http://
peterslab.org/MSTools/).
3 Methods
3.1 Verification
of Protein–DNA
Complex Formation by
a Gel Shift Assay
Prior to the HDX-MS experiment, it is advisable to verify whether
the DNA duplex is present and that the complex between protein
and ligand (here DNA) is indeed formed upon mixing of the
components. In this respect, knowledge of the binding constants
offers an advantage as the experimental setup can be adjusted to
achieve full protein occupancy (complex presence) during the labeling step [18]. Here, we use gel shift assay to check DNA duplex
formation and its binding to the studied protein.
1. Dilute both DNA strands in LC-MS purity water and mix them
at equimolar ratio.
2. Heat the mixture at 95
C for 1 min, and let it slowly cool
down to room temperature.
3. Dilute/buffer exchange the protein into a buffer suitable for
the complex which is later used for H/D exchange (20 mM
HEPES, pH 7.4, 150 mM NaCl, see Note 5) to a concentration at which full occupancy of the protein by DNA is achieved
(see Note 6).
4. Add equimolar amounts of dsDNA to the protein and let the
mixture equilibrate for at least 2 min at a temperature where
both the dsDNA and protein are stable (see Note 7).
5. Prepare following samples: 200 ng of both single-stranded
oligonucleotides, 200 ng of free dsDNA, 5 μg of free protein,
and the equimolar protein–DNA complex equal to 5 μg of the
protein (see Note 8). All components must be prepared in the
same buffer.
6. Add loading buffer to all samples.
7. Prepare non-denaturing polyacrylamide gel by mixing all components except APS. Add APS just before pouring the mixture
into a gel casting tray (see Note 9).
HDX-MS of Protein-DNA Complexes
197
27. LC solvent B (0.4% formic acid, 95% acetonitrile, 4.6% water).
28. Electrospray ionization (ESI) mass spectrometer (any
ESI-equipped MS with reasonable resolution is usable; in our
case, we used an electrospray ionization—Fourier transform
ion cyclotron resonance 15T solariX XR, Bruker Daltonics).
29. Software: DataAnalysis 4.1 (Bruker Daltonics), ProteinScape 4
(Bruker Daltonics), DeutEx (http://peterslab.org/downloads.
php under HDX tools section), mMass (http://mmass.org),
PyMol (https://pymol.org/; v 2.1.0), MSTools (http://
peterslab.org/MSTools/).
3 Methods
3.1 Verification
of Protein–DNA
Complex Formation by
a Gel Shift Assay
Prior to the HDX-MS experiment, it is advisable to verify whether
the DNA duplex is present and that the complex between protein
and ligand (here DNA) is indeed formed upon mixing of the
components. In this respect, knowledge of the binding constants
offers an advantage as the experimental setup can be adjusted to
achieve full protein occupancy (complex presence) during the labeling step [18]. Here, we use gel shift assay to check DNA duplex
formation and its binding to the studied protein.
1. Dilute both DNA strands in LC-MS purity water and mix them
at equimolar ratio.
2. Heat the mixture at 95
C for 1 min, and let it slowly cool
down to room temperature.
3. Dilute/buffer exchange the protein into a buffer suitable for
the complex which is later used for H/D exchange (20 mM
HEPES, pH 7.4, 150 mM NaCl, see Note 5) to a concentration at which full occupancy of the protein by DNA is achieved
(see Note 6).
4. Add equimolar amounts of dsDNA to the protein and let the
mixture equilibrate for at least 2 min at a temperature where
both the dsDNA and protein are stable (see Note 7).
5. Prepare following samples: 200 ng of both single-stranded
oligonucleotides, 200 ng of free dsDNA, 5 μg of free protein,
and the equimolar protein–DNA complex equal to 5 μg of the
protein (see Note 8). All components must be prepared in the
same buffer.
6. Add loading buffer to all samples.
7. Prepare non-denaturing polyacrylamide gel by mixing all components except APS. Add APS just before pouring the mixture
into a gel casting tray (see Note 9).
HDX-MS of Protein-DNA Complexes
197