hydrochloride was replaced by urea, and an alternative aspartic
protease was used in the quench buffer [12].
In this study, drawing upon the experience gained through our
own work, we present an experimental protocol for the characterization of protein interactions with DNA (in the form of an oligonucleotide) as used in our group. We have repeatedly observed that
careful optimization and proper control over the quality of the
DNA entering an HDX experiment (amount of dsDNA formed,
purity, salt concentration) are crucial for boosting its ability to bind
to the interaction partner. Ensuring as strong an interaction as
possible in this manner then sometimes largely mitigates the problems stemming from the DNA presence, as the amount of oligonucleotides used can be kept to a minimum (optimally down to 1:1
molar ratio to the protein), while still providing virtually complete
saturation of the transcription factor (TF). Sometimes, however,
even the best quality of sample is not enough, and the aforementioned techniques for coping with DNA come into play.
In the following set of protocols, we describe ways to perform
protein–DNA HDX experiments, together with some of the potential pitfalls, with emphasis on the interaction of transcription factors
with their DNA-response elements. As we have observed that
protein:DNA systems can behave surprisingly differently even
when the proteins as well as DNAs used are similar in size, we will
be using two example systems. Forkhead box protein O4
(FOXO4)/DAF16 [13, 14] serves as an example where short
dsDNA (13 bp) does not interfere significantly with peptide recovery, and only digestion conditions are optimized to reach good
HDX spatial resolution. On the other hand, the TEA domain
family member 1 (TEAD1)/M-CAT [15] represents the opposite
situation when a 15-bp long oligonucleotide has a strong impact on
peptide recovery. These striking differences in behavior stress the
importance of having a vast array of conditions, proteases, and
technical tricks at one’s disposal to fine-tune the HDX protocol.
The procedures we use, as described herein, should provide prospective users with a set of tools to successfully optimize conditions
for HDX and perform their own analyses of a range of TF–DNA
complexes.
2 Materials
1. Protein to be analyzed (see Note 2).
2. Forward and reverse DNA strands (see Note 3).
3. Buffer suitable for the complex (e.g., 20 mM HEPES, pH 7.4,
150 mM NaCl).
4. Gel casting tray and electrophoresis apparatus (Bio-Rad).
HDX-MS of Protein-DNA Complexes
195
protease was used in the quench buffer [12].
In this study, drawing upon the experience gained through our
own work, we present an experimental protocol for the characterization of protein interactions with DNA (in the form of an oligonucleotide) as used in our group. We have repeatedly observed that
careful optimization and proper control over the quality of the
DNA entering an HDX experiment (amount of dsDNA formed,
purity, salt concentration) are crucial for boosting its ability to bind
to the interaction partner. Ensuring as strong an interaction as
possible in this manner then sometimes largely mitigates the problems stemming from the DNA presence, as the amount of oligonucleotides used can be kept to a minimum (optimally down to 1:1
molar ratio to the protein), while still providing virtually complete
saturation of the transcription factor (TF). Sometimes, however,
even the best quality of sample is not enough, and the aforementioned techniques for coping with DNA come into play.
In the following set of protocols, we describe ways to perform
protein–DNA HDX experiments, together with some of the potential pitfalls, with emphasis on the interaction of transcription factors
with their DNA-response elements. As we have observed that
protein:DNA systems can behave surprisingly differently even
when the proteins as well as DNAs used are similar in size, we will
be using two example systems. Forkhead box protein O4
(FOXO4)/DAF16 [13, 14] serves as an example where short
dsDNA (13 bp) does not interfere significantly with peptide recovery, and only digestion conditions are optimized to reach good
HDX spatial resolution. On the other hand, the TEA domain
family member 1 (TEAD1)/M-CAT [15] represents the opposite
situation when a 15-bp long oligonucleotide has a strong impact on
peptide recovery. These striking differences in behavior stress the
importance of having a vast array of conditions, proteases, and
technical tricks at one’s disposal to fine-tune the HDX protocol.
The procedures we use, as described herein, should provide prospective users with a set of tools to successfully optimize conditions
for HDX and perform their own analyses of a range of TF–DNA
complexes.
2 Materials
1. Protein to be analyzed (see Note 2).
2. Forward and reverse DNA strands (see Note 3).
3. Buffer suitable for the complex (e.g., 20 mM HEPES, pH 7.4,
150 mM NaCl).
4. Gel casting tray and electrophoresis apparatus (Bio-Rad).
HDX-MS of Protein-DNA Complexes
195