most useful reducing agent is the acid functional compound
Tris(2-carboxyethyl)phosphine (TCEP) [34, 40]. The
exchange rate of amides with deuterium is an acid–base catalyzed process, with a global minimum at approx. pH 2.5 [41];
therefore, the quench buffer should be at this pH. The concentration of acid in the quench buffer will depend of the
buffering capacity of the D 2 O buffer, which generally contains
Tris or HEPES and the protein buffer(s). That being said, one
must check the pH of a mock sample containing all the components in the HDX reaction without protein and D 2 O (i.e., a
solution containing the exact volumes of quench solution,
protein buffer(s), and H 2 O buffer) before running any samples
on the mass spectrometer as to avoid inactivating or ruining the
pepsin columns (pepsin becomes inactive at pH >6.5 and fully
denatures at pH 8.0). Additionally, the quenching solution is
kept cold (~1
C) throughout the HDX setup to minimize back
exchange by dramatically slowing the kinetics of the exchange
reaction.
5. Successful HDX experiments heavily depend on accuracy, specificity, and repeatability of sample preparation. Consistency in
technique when mixing the D 2 O or quench with the sample
and when flash freezing the protein in liquid nitrogen will
reduce variability in deuterium incorporation between replicates. Additionally, it is important to maintain constant temperature (experiments should be carried out in a climate
controlled room) as well as pH throughout the setup and
across the various conditions. All comparisons between conditions must use identical buffers. For example, if the protein of
interest is in buffer A and its binding partner is in buffer B, all
HDX samples should contain the same volume of buffer A and
B regardless of whether the protein and/or its binding partner
is present in that sample.
6. A consequence of having multiple columns incorporated in the
fluidics system is an increased probability of carryover between
samples. The amount of carryover will be dependent on the
specific protein and the amount being run on the system. As
previously mentioned, one of the steps taken to mitigate this
issue is to start the system up with a ~20-min blank and a ~8min short blank to elute peptides that carried over from the
previous day. Peptides can also carryover between samples;
therefore, to reduce this occurrence, a short ~8-min blank is
run between samples. Additionally, every method includes a
sample loop/pepsin washing step that occurs simultaneously
while peptides elute from the trap. This step is automated using
the LEAP PAL liquid handling robot which first injects Sample
Loop/Pepsin Wash Buffer into the sample loop and then puts
the sample loop in line with the pepsin columns. From there,
482
Jordan T. B. Stariha et al.
Tris(2-carboxyethyl)phosphine (TCEP) [34, 40]. The
exchange rate of amides with deuterium is an acid–base catalyzed process, with a global minimum at approx. pH 2.5 [41];
therefore, the quench buffer should be at this pH. The concentration of acid in the quench buffer will depend of the
buffering capacity of the D 2 O buffer, which generally contains
Tris or HEPES and the protein buffer(s). That being said, one
must check the pH of a mock sample containing all the components in the HDX reaction without protein and D 2 O (i.e., a
solution containing the exact volumes of quench solution,
protein buffer(s), and H 2 O buffer) before running any samples
on the mass spectrometer as to avoid inactivating or ruining the
pepsin columns (pepsin becomes inactive at pH >6.5 and fully
denatures at pH 8.0). Additionally, the quenching solution is
kept cold (~1
C) throughout the HDX setup to minimize back
exchange by dramatically slowing the kinetics of the exchange
reaction.
5. Successful HDX experiments heavily depend on accuracy, specificity, and repeatability of sample preparation. Consistency in
technique when mixing the D 2 O or quench with the sample
and when flash freezing the protein in liquid nitrogen will
reduce variability in deuterium incorporation between replicates. Additionally, it is important to maintain constant temperature (experiments should be carried out in a climate
controlled room) as well as pH throughout the setup and
across the various conditions. All comparisons between conditions must use identical buffers. For example, if the protein of
interest is in buffer A and its binding partner is in buffer B, all
HDX samples should contain the same volume of buffer A and
B regardless of whether the protein and/or its binding partner
is present in that sample.
6. A consequence of having multiple columns incorporated in the
fluidics system is an increased probability of carryover between
samples. The amount of carryover will be dependent on the
specific protein and the amount being run on the system. As
previously mentioned, one of the steps taken to mitigate this
issue is to start the system up with a ~20-min blank and a ~8min short blank to elute peptides that carried over from the
previous day. Peptides can also carryover between samples;
therefore, to reduce this occurrence, a short ~8-min blank is
run between samples. Additionally, every method includes a
sample loop/pepsin washing step that occurs simultaneously
while peptides elute from the trap. This step is automated using
the LEAP PAL liquid handling robot which first injects Sample
Loop/Pepsin Wash Buffer into the sample loop and then puts
the sample loop in line with the pepsin columns. From there,
482
Jordan T. B. Stariha et al.