non-denaturing deuterated buffer. This will lower the denaturant concentration which can alter the subsequent workflow
outcomes. From this perspective, using urea is the better
option (see Subheading 3.2). Quench the reaction and eventually freeze the sample, if this step is included in the workflow.
3.4 Mass
Spectrometric
Analysis of Deuterated
Samples
1. Use the LC system as in Subheading 3.2 and perform steps
2–4. Start all LC pumps, calibrate the mass spectrometer,
pre-wash, and pre-condition the protease column. Then run
the standard protein followed by two blank injections (see
Note 28).
2. Before the end of previous analysis, take one of the partially
deuterated samples from the freezer and start thawing
it. Depending on the quench buffer composition, it will melt
in 30 s to 1 min. As soon as it is thawed, inject it for analysis.
3. Repeat this for all collected samples (see Note 29).
4. Analyze fully deuterated control exactly as the partially deuterated samples.
5. Finally run two blanks followed by non-deuterated samples for
each experimental condition.
6. Export and/or pre-process all LC-MS acquired data and prepare them for final data processing.
3.5 Interpreting Data
from H/D Exchange
Nowadays, there are several different programs that are capable of
largely automatic HDX-MS data processing. Waters users rely on
their DynamX suite while others can use HDExaminer from Sierra
Analytics that supports all native file types as well as open source
ones. Another option for Waters and Thermo users is represented
by the HDX Workbench [31] from Omics Informatics. Alternatively, there are software tools freely available from different
research groups [22, 32–35]. These and other programs and their
workflows were recently reviewed in detail by Claesen and Burzykowski and also by Eggertson et al. which in addition offers another
view on HDX-MS data processing [36, 37].
The basic principle which is crucial to the understanding of the
workflow and its requirements is, however, the same. To make this
protocol widely applicable, we will show two possible scenarios.
One, relying on a manual interpretation which is nowadays outdated and extremely laborious. However, it demonstrates well the
basic principle which the available programs automate. It can also
be useful for validation purposes and in specific cases (extraction of
EX1/EX2 data). The other workflow presented here employs our
own software called DeutEx (see Note 30).
Data processing in the HDX-MS workflow consists of four
major steps. First is the identification of peptides generated during
the proteolysis step and their temporal localization within an LC
206
Ruzena Filandrova et al.
outcomes. From this perspective, using urea is the better
option (see Subheading 3.2). Quench the reaction and eventually freeze the sample, if this step is included in the workflow.
3.4 Mass
Spectrometric
Analysis of Deuterated
Samples
1. Use the LC system as in Subheading 3.2 and perform steps
2–4. Start all LC pumps, calibrate the mass spectrometer,
pre-wash, and pre-condition the protease column. Then run
the standard protein followed by two blank injections (see
Note 28).
2. Before the end of previous analysis, take one of the partially
deuterated samples from the freezer and start thawing
it. Depending on the quench buffer composition, it will melt
in 30 s to 1 min. As soon as it is thawed, inject it for analysis.
3. Repeat this for all collected samples (see Note 29).
4. Analyze fully deuterated control exactly as the partially deuterated samples.
5. Finally run two blanks followed by non-deuterated samples for
each experimental condition.
6. Export and/or pre-process all LC-MS acquired data and prepare them for final data processing.
3.5 Interpreting Data
from H/D Exchange
Nowadays, there are several different programs that are capable of
largely automatic HDX-MS data processing. Waters users rely on
their DynamX suite while others can use HDExaminer from Sierra
Analytics that supports all native file types as well as open source
ones. Another option for Waters and Thermo users is represented
by the HDX Workbench [31] from Omics Informatics. Alternatively, there are software tools freely available from different
research groups [22, 32–35]. These and other programs and their
workflows were recently reviewed in detail by Claesen and Burzykowski and also by Eggertson et al. which in addition offers another
view on HDX-MS data processing [36, 37].
The basic principle which is crucial to the understanding of the
workflow and its requirements is, however, the same. To make this
protocol widely applicable, we will show two possible scenarios.
One, relying on a manual interpretation which is nowadays outdated and extremely laborious. However, it demonstrates well the
basic principle which the available programs automate. It can also
be useful for validation purposes and in specific cases (extraction of
EX1/EX2 data). The other workflow presented here employs our
own software called DeutEx (see Note 30).
Data processing in the HDX-MS workflow consists of four
major steps. First is the identification of peptides generated during
the proteolysis step and their temporal localization within an LC
206
Ruzena Filandrova et al.