Starting from the moment of quenching and throughout the
digestion, desalting, and subsequent analysis, the sample is exposed
to protonated solvents. Even though the minimal exchange conditions are maintained through these steps, the sample still undergoes
deuterium loss. The level of back-exchange is not uniform and
depends on the sequence of individual peptides. However, this
loss can be measured by analyzing a fully deuterated sample and
then using these data, to correct deuteration levels on partially
deuterated peptides [28]. In a typical measurement comparing
two protein states, relative deuterium levels are the desired measure, and there is no need for back-exchange correction. Thus, in
these cases, this control is often omitted. This is also likely due to
the fact that no clear and universal protocol exists and that proteins
often tend to precipitate or degrade before their full deuteration is
achieved. However, the knowledge of the actual back-exchange is
needed when absolute levels of deuteration are sought (e.g., in
cases where mutated sequences of the same protein are
compared) [30].
1. Prepare the required number of 0.5-ml Eppendorf tubes (for
two conditions, three labeling replicates, and six time points,
this would mean 36 tubes in total) with quench solution—
50 μl of 500 mM glycine-Cl buffer, pH 2.3 (see Note 22).
2. Prepare (bigger) tubes with the protein alone and with the
pre-formed protein–DNA complex. In these, the ten-fold dilution into deuterated buffer and the labeling reaction will be
performed. At six preselected time points (see Note 23), 50 μl
aliquots will be removed from the reaction and quenched. As
three labeling replicates are to be done, three tubes with protein alone and three with the protein–DNA complex, each
containing 40 μl of 2 μM protein solution must be prepared
(see Note 24).
3. Prepare 2 ml of deuterated buffer (20 mM HEPES, 150 mM
NaCl, pD 7.4) (see Note 25).
4. Prepare a time schedule that allows efficient pipetting especially
in cases where larger amount of conditions or direct technical
(labeling) replicates are performed at once. This can be easily
done using MSTools script “Experiment planner” (http://
peterslab.org/MSTools/HDExpPlanner/HDExpPlanner.
php). For two experimental conditions, each replicated three
times, and aliquot collection at 20 s, 2 min, 5 min, 20 min, 1 h,
3 h, this may look as shown in Fig. 4.
5. Prepare automatic pipettes with pre-set volumes for H/D mixing (360 μl) and aliquot collection (50 μl).
6. Use a timer that allows countdown followed by a count-up. Set
20 s count-down and start it. Aspirate 360 μl of the deuterated
buffer and when the timer reaches zero (start of the whole
204
Ruzena Filandrova et al.
digestion, desalting, and subsequent analysis, the sample is exposed
to protonated solvents. Even though the minimal exchange conditions are maintained through these steps, the sample still undergoes
deuterium loss. The level of back-exchange is not uniform and
depends on the sequence of individual peptides. However, this
loss can be measured by analyzing a fully deuterated sample and
then using these data, to correct deuteration levels on partially
deuterated peptides [28]. In a typical measurement comparing
two protein states, relative deuterium levels are the desired measure, and there is no need for back-exchange correction. Thus, in
these cases, this control is often omitted. This is also likely due to
the fact that no clear and universal protocol exists and that proteins
often tend to precipitate or degrade before their full deuteration is
achieved. However, the knowledge of the actual back-exchange is
needed when absolute levels of deuteration are sought (e.g., in
cases where mutated sequences of the same protein are
compared) [30].
1. Prepare the required number of 0.5-ml Eppendorf tubes (for
two conditions, three labeling replicates, and six time points,
this would mean 36 tubes in total) with quench solution—
50 μl of 500 mM glycine-Cl buffer, pH 2.3 (see Note 22).
2. Prepare (bigger) tubes with the protein alone and with the
pre-formed protein–DNA complex. In these, the ten-fold dilution into deuterated buffer and the labeling reaction will be
performed. At six preselected time points (see Note 23), 50 μl
aliquots will be removed from the reaction and quenched. As
three labeling replicates are to be done, three tubes with protein alone and three with the protein–DNA complex, each
containing 40 μl of 2 μM protein solution must be prepared
(see Note 24).
3. Prepare 2 ml of deuterated buffer (20 mM HEPES, 150 mM
NaCl, pD 7.4) (see Note 25).
4. Prepare a time schedule that allows efficient pipetting especially
in cases where larger amount of conditions or direct technical
(labeling) replicates are performed at once. This can be easily
done using MSTools script “Experiment planner” (http://
peterslab.org/MSTools/HDExpPlanner/HDExpPlanner.
php). For two experimental conditions, each replicated three
times, and aliquot collection at 20 s, 2 min, 5 min, 20 min, 1 h,
3 h, this may look as shown in Fig. 4.
5. Prepare automatic pipettes with pre-set volumes for H/D mixing (360 μl) and aliquot collection (50 μl).
6. Use a timer that allows countdown followed by a count-up. Set
20 s count-down and start it. Aspirate 360 μl of the deuterated
buffer and when the timer reaches zero (start of the whole
204
Ruzena Filandrova et al.