script at the MSTools website (http://peterslab.org/
MSTools/DrawMap/DrawMap.php) and eventually use
Excel macro Digestion_metrics.xlsm (http://peterslab.org/
downloads.php under HDX tools section) to extract the digestion metrics.
11. Based on the peptide length, redundancy, coverage, and number of gaps in the peptide map, try optimizing the digestion by
using alternative proteases or quench buffer additives as
described above (example in Fig. 3) (see Note 19).
3.3 Hydrogen/
Deuterium Exchange
The methodology described here represents a manual approach.
Even though nowadays there are robotic solutions available that
can do the sample preparation and injection automatically, they are
quite specific depending on the manufacturer of the robot and the
software controlling it. The manual approach represents a straightforward and inexpensive way that can be easily set up in any laboratory possessing an LC system and a mass spectrometer (see Note
20).
The actual H/D exchange should be started from equilibrated
solutions in which the complex is fully formed, and the temperature
of all components is stable. The first step in the deuterium labeling
reaction is the dilution of the studied protein into a deuterated
buffer (see Note 21) that has otherwise identical composition as the
initial buffer used for complex pre-formation. After a predetermined time, the exchange is “stopped” by the addition of acidic
quench buffer and by lowering the temperature or eventually by
flash freezing in liquid nitrogen.
The experiments should ideally be done (at least) in triplicate to
allow for statistical validation. There are several ways how to perform the replication in HDX-MS. The highest validity is achieved
by doing a biological replication (in the case of recombinant proteins this means using independent protein production batches). A
less significant (though commonly used) level of replication (technical) is reached by separate deuterium labeling, and the lowest tier
is represented by measuring the same labeling reaction repeatedly
[28]. Replication also allows one to estimate the significance level
above which the differences in deuteration between individual
states may be considered meaningful [29].
ä
Fig. 3 (continued) coverage but lower spatial resolution—on average it had longest peptides. Nepenthesin2 (red bars) led to over-digestion, which is indicated by gaps in the sequence, and the identified peptides are
mostly short ones. High activity of the protease resulted in the generation of short peptides and incomplete
sequence coverage. The green bars show the final conditions where aspergillopepsin (protease type XIII) was
utilized in the presence of urea. Addition of denaturing agent was necessary as the protein in the presence of
oligonucleotides precipitated. Addition of urea (optimized to final 2 M concentration) led to much better protein
recovery. In both examples shown here, the advantage of alternative proteases for the digestion of DNA
binding proteins (which are rich in basic residues) is demonstrated through their ability to cleave after the Lys
and Arg residues (highlighted by arrowheads above the sequence)
HDX-MS of Protein-DNA Complexes
203
MSTools/DrawMap/DrawMap.php) and eventually use
Excel macro Digestion_metrics.xlsm (http://peterslab.org/
downloads.php under HDX tools section) to extract the digestion metrics.
11. Based on the peptide length, redundancy, coverage, and number of gaps in the peptide map, try optimizing the digestion by
using alternative proteases or quench buffer additives as
described above (example in Fig. 3) (see Note 19).
3.3 Hydrogen/
Deuterium Exchange
The methodology described here represents a manual approach.
Even though nowadays there are robotic solutions available that
can do the sample preparation and injection automatically, they are
quite specific depending on the manufacturer of the robot and the
software controlling it. The manual approach represents a straightforward and inexpensive way that can be easily set up in any laboratory possessing an LC system and a mass spectrometer (see Note
20).
The actual H/D exchange should be started from equilibrated
solutions in which the complex is fully formed, and the temperature
of all components is stable. The first step in the deuterium labeling
reaction is the dilution of the studied protein into a deuterated
buffer (see Note 21) that has otherwise identical composition as the
initial buffer used for complex pre-formation. After a predetermined time, the exchange is “stopped” by the addition of acidic
quench buffer and by lowering the temperature or eventually by
flash freezing in liquid nitrogen.
The experiments should ideally be done (at least) in triplicate to
allow for statistical validation. There are several ways how to perform the replication in HDX-MS. The highest validity is achieved
by doing a biological replication (in the case of recombinant proteins this means using independent protein production batches). A
less significant (though commonly used) level of replication (technical) is reached by separate deuterium labeling, and the lowest tier
is represented by measuring the same labeling reaction repeatedly
[28]. Replication also allows one to estimate the significance level
above which the differences in deuteration between individual
states may be considered meaningful [29].
ä
Fig. 3 (continued) coverage but lower spatial resolution—on average it had longest peptides. Nepenthesin2 (red bars) led to over-digestion, which is indicated by gaps in the sequence, and the identified peptides are
mostly short ones. High activity of the protease resulted in the generation of short peptides and incomplete
sequence coverage. The green bars show the final conditions where aspergillopepsin (protease type XIII) was
utilized in the presence of urea. Addition of denaturing agent was necessary as the protein in the presence of
oligonucleotides precipitated. Addition of urea (optimized to final 2 M concentration) led to much better protein
recovery. In both examples shown here, the advantage of alternative proteases for the digestion of DNA
binding proteins (which are rich in basic residues) is demonstrated through their ability to cleave after the Lys
and Arg residues (highlighted by arrowheads above the sequence)
HDX-MS of Protein-DNA Complexes
203