(i.e., samples with single protein and no lipid), add the same
volume of lipid buffer as you would lipid to the lipidcontaining samples to ensure all samples are in the same buffer
(see Note 5).
5. Add 40 μL D 2 O buffer to start the exchange reaction.
6. Incubate the reaction at 20
C for the correct amount of time
(either 3, 30, 300, or 3000 s). For the 3000 s time point, the
sample is purged with nitrogen and sealed with parafilm to
minimize any problems with oxidation or D 2 O exchange with
H 2 O in ambient air.
7. Add 20 μL quench solution to terminate the exchange reaction. To achieve the 3 s exposures, have the quench volume
already drawn up in a pipette. Add the D 2 O, mix a specific
number of times, immediately add quench using the second
pipette, mix a specific number of times, and flash freeze in
liquid N 2 .
8. Immediately flash freeze the sample tube in liquid N 2 following
addition of quench.
9. Make non-deuterated samples in the same fashion but instead
of adding D 2 O buffer, add H 2 O buffer. “Quench” immediately (no exchange reaction occurs in H 2 O buffer; therefore no
incubation time is required) and flash freeze tube in liquid N 2 .
10. Store all samples at À80
C. Flash frozen samples can be stored
for up to a month at this temperature without affecting the
amide exchange.
3.2 Operating
the Mass
Spectrometer
3.2.1 Precautions
Necessary When Starting
a Project
The success of an HDX experiment is highly dependent on taking
proper and regular care of the instruments. This includes the
following:
1. Buffer levels. All buffers (Mobile Phase, Needle Wash, and
Digestion Buffer) used for the LC and RTC PAL fluidics
systems must be monitored at all times to avoid running the
system dry during an experiment, thus risking the loss of data as
well as equipment damage. Buffers should be changed at least
every 3 months.
2. LC purging. Air bubbles can accumulate within the LC pump
system if it is not used for more than a few days, therefore
risking pumping air onto the trap and analytical columns. To
avoid doing so, the LC fluidics system must be purged, which
involves passing Mobile Phases A and B through the pump
system into waste at a high flow rate for a period of at least
10 min. This can be done using the liquid chromatography
control software, in this case “Chromeleon.”
3. Mass spectrometer calibration. To ensure maximum accuracy,
the mass spectrometer must be calibrated at least twice a week
Probing Protein-Membrane Interactions Using HDX-MS
473
volume of lipid buffer as you would lipid to the lipidcontaining samples to ensure all samples are in the same buffer
(see Note 5).
5. Add 40 μL D 2 O buffer to start the exchange reaction.
6. Incubate the reaction at 20
C for the correct amount of time
(either 3, 30, 300, or 3000 s). For the 3000 s time point, the
sample is purged with nitrogen and sealed with parafilm to
minimize any problems with oxidation or D 2 O exchange with
H 2 O in ambient air.
7. Add 20 μL quench solution to terminate the exchange reaction. To achieve the 3 s exposures, have the quench volume
already drawn up in a pipette. Add the D 2 O, mix a specific
number of times, immediately add quench using the second
pipette, mix a specific number of times, and flash freeze in
liquid N 2 .
8. Immediately flash freeze the sample tube in liquid N 2 following
addition of quench.
9. Make non-deuterated samples in the same fashion but instead
of adding D 2 O buffer, add H 2 O buffer. “Quench” immediately (no exchange reaction occurs in H 2 O buffer; therefore no
incubation time is required) and flash freeze tube in liquid N 2 .
10. Store all samples at À80
C. Flash frozen samples can be stored
for up to a month at this temperature without affecting the
amide exchange.
3.2 Operating
the Mass
Spectrometer
3.2.1 Precautions
Necessary When Starting
a Project
The success of an HDX experiment is highly dependent on taking
proper and regular care of the instruments. This includes the
following:
1. Buffer levels. All buffers (Mobile Phase, Needle Wash, and
Digestion Buffer) used for the LC and RTC PAL fluidics
systems must be monitored at all times to avoid running the
system dry during an experiment, thus risking the loss of data as
well as equipment damage. Buffers should be changed at least
every 3 months.
2. LC purging. Air bubbles can accumulate within the LC pump
system if it is not used for more than a few days, therefore
risking pumping air onto the trap and analytical columns. To
avoid doing so, the LC fluidics system must be purged, which
involves passing Mobile Phases A and B through the pump
system into waste at a high flow rate for a period of at least
10 min. This can be done using the liquid chromatography
control software, in this case “Chromeleon.”
3. Mass spectrometer calibration. To ensure maximum accuracy,
the mass spectrometer must be calibrated at least twice a week
Probing Protein-Membrane Interactions Using HDX-MS
473