2.2 HDX-MS Setup
Buffers
1. Quench buffer: 2 M ! 99% purity guanidine +3% LC/MS grade
formic acid (FA). Measure out 9.553 g of !99% purity guanidine hydrochloride into a detergent-free vessel and bring the
total volume up to 32.3 mL. Add 1 mL of 100% FA to bring
the total volume to 33.3 mL (final FA concentration of 3%).
Keep buffer on ice at all times during the experiment as
quenching must be completed at a low temperature (~1
C)
to minimize back exchange.
2. D 2 O buffer: 10 mM !99.5% purity HEPES (pH 7.5), 100 mM
!99% purity NaCl, 98% D 2 O. This buffer needs to be made
fresh for each experiment to prevent exchange with H 2 O present in ambient air. For making 1 mL of D 2 O buffer, combine
20 μL of 5 M NaCl with 20 μL of 0.5 M HEPES (pH 7.5) in a
1.5 mL microcentrifuge tube prior to opening the D 2 O. Crack
open an ampule of D 2 O and use a glass pipette to rapidly
transfer the D 2 O into the 1.5 mL microcentrifuge tube containing the NaCl and HEPES. Close the tube immediately after
addition of D 2 O and open only as needed to reduce the
amount of contact with H 2 O from the environment. Mix by
inverting the tube several times. The buffer should be equilibrated to the temperature planned in the H/D exchange
experiment.
3. H 2 O buffer: 10 mM !99.5% purity HEPES (pH 7.5), 100 mM
!99% purity NaCl. For making 1 mL of H 2 O buffer, combine
20 μL of 5 M NaCl with 20 μL of 0.5 M HEPES (pH 7.5) in a
1.5 mL microcentrifuge tube. Add 1 mL of deionized water to
the tube and mix well. Keep the buffer in the same conditions
as the D 2 O buffer. As with the D 2 O buffer, remake for each
experiment.
4. Lipid buffer: !99% purity 100 mM KCl, !99.5% purity 20 mM
HEPES (pH 7.5). Combine 1 mL of 5 M KCl and 2 mL of
0.5 M HEPES (pH 7.5). Bring up to a final volume of 50 mL
with deionized water and filter through a 0.22 μm filter.
5. Protein buffer: The protein buffer used in the exchange reaction will be the same buffer used in the final purification stage
for the protein (i.e., the final storage buffer). For the best
results, save and store some of the buffer used during purification. It is important to make sure the buffer does not contain
any detergent. If multiple proteins are being assessed, and their
buffers are different, both buffers will need to be used.
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Jordan T. B. Stariha et al.
Buffers
1. Quench buffer: 2 M ! 99% purity guanidine +3% LC/MS grade
formic acid (FA). Measure out 9.553 g of !99% purity guanidine hydrochloride into a detergent-free vessel and bring the
total volume up to 32.3 mL. Add 1 mL of 100% FA to bring
the total volume to 33.3 mL (final FA concentration of 3%).
Keep buffer on ice at all times during the experiment as
quenching must be completed at a low temperature (~1
C)
to minimize back exchange.
2. D 2 O buffer: 10 mM !99.5% purity HEPES (pH 7.5), 100 mM
!99% purity NaCl, 98% D 2 O. This buffer needs to be made
fresh for each experiment to prevent exchange with H 2 O present in ambient air. For making 1 mL of D 2 O buffer, combine
20 μL of 5 M NaCl with 20 μL of 0.5 M HEPES (pH 7.5) in a
1.5 mL microcentrifuge tube prior to opening the D 2 O. Crack
open an ampule of D 2 O and use a glass pipette to rapidly
transfer the D 2 O into the 1.5 mL microcentrifuge tube containing the NaCl and HEPES. Close the tube immediately after
addition of D 2 O and open only as needed to reduce the
amount of contact with H 2 O from the environment. Mix by
inverting the tube several times. The buffer should be equilibrated to the temperature planned in the H/D exchange
experiment.
3. H 2 O buffer: 10 mM !99.5% purity HEPES (pH 7.5), 100 mM
!99% purity NaCl. For making 1 mL of H 2 O buffer, combine
20 μL of 5 M NaCl with 20 μL of 0.5 M HEPES (pH 7.5) in a
1.5 mL microcentrifuge tube. Add 1 mL of deionized water to
the tube and mix well. Keep the buffer in the same conditions
as the D 2 O buffer. As with the D 2 O buffer, remake for each
experiment.
4. Lipid buffer: !99% purity 100 mM KCl, !99.5% purity 20 mM
HEPES (pH 7.5). Combine 1 mL of 5 M KCl and 2 mL of
0.5 M HEPES (pH 7.5). Bring up to a final volume of 50 mL
with deionized water and filter through a 0.22 μm filter.
5. Protein buffer: The protein buffer used in the exchange reaction will be the same buffer used in the final purification stage
for the protein (i.e., the final storage buffer). For the best
results, save and store some of the buffer used during purification. It is important to make sure the buffer does not contain
any detergent. If multiple proteins are being assessed, and their
buffers are different, both buffers will need to be used.
468
Jordan T. B. Stariha et al.