2. Determine the reaction volume and the final concentration of
the protein(s) and lipid(s) that will be used. The concentrations
must be carefully optimized to achieve the desired amount of
protein in each sample to maximize sensitivity, while at the
same time limiting carryover, as well as maximize occupancy
and percent D 2 O present in the exchange reaction (see Notes
2 and 3). Experiments on Akt were carried out at a final protein
amount of 20 pmol per sample, in a final volume of 50 μL. The
final protein concentration of Akt was 400 nM, and the final
concentration of lipid vesicles (20% cholesterol, 30% PC, 15%
PS, 35% PE, and 5% PIP 3 ) was 400 μM. Lipid vesicles were
generated by extrusion through 0.1 μm filters in lipid buffer.
3. Calculate the final concentration of D 2 O in the exchange reaction. In the Akt experiment, the final concentration of D 2 O was
78%.
4. Determine the quenching conditions that mediate digestion
and denaturation (see Note 4). The quenching conditions used
in the Akt experiment were 20 μL of ice cold 2 M guanidine
HCl, 3% formic acid, giving a final concentration of 0.6 M
GdHCl and 0.8% formic acid.
5. Determine which D 2 O exposure time points you want to
assess. Common time points used are 3 s, 30 s, 300 s, and
3000 s exposures, which enable probing a range of amide
hydrogen exchange rates. If this range is not sufficient to fully
sample the spectrum of amide exchange rates, different temperatures and pH values can be used [35].
3.1.2 Execution of HDX
1. Thaw the appropriate amount of protein and lipids for entire
experiment. Once thawed, keep protein on ice and lipids at
20
C.
2. Mix protein well and spin down in a cooled tabletop centrifuge
(4
C) at 15,000 rpm for 5 min to remove any aggregate. Make
sure to check the protein concentrations prior to any experimentation as the protein may not be at the same concentration
that it was originally stored at. Adjust calculations as necessary
to ensure final protein concentrations are correct.
3. Label and organize 0.6 mL Eppendorf tubes for all samples
(indicate condition and time point on label). It is ideal to have a
triplicate set of all exchange reactions. Make sure to include
tubes for at least two non-deuterated apo samples. One will be
used to identify the peptides generated during proteolysis (see
Subheading 3.3.1); the other will be used as a non-deuterated
reference that the analytic software will compare the deuterated
samples to.
4. Combine both protein and lipid components in a final volume
of 10 μL and incubate at 20
C for 2 min. For the apo samples
472
Jordan T. B. Stariha et al.
the protein(s) and lipid(s) that will be used. The concentrations
must be carefully optimized to achieve the desired amount of
protein in each sample to maximize sensitivity, while at the
same time limiting carryover, as well as maximize occupancy
and percent D 2 O present in the exchange reaction (see Notes
2 and 3). Experiments on Akt were carried out at a final protein
amount of 20 pmol per sample, in a final volume of 50 μL. The
final protein concentration of Akt was 400 nM, and the final
concentration of lipid vesicles (20% cholesterol, 30% PC, 15%
PS, 35% PE, and 5% PIP 3 ) was 400 μM. Lipid vesicles were
generated by extrusion through 0.1 μm filters in lipid buffer.
3. Calculate the final concentration of D 2 O in the exchange reaction. In the Akt experiment, the final concentration of D 2 O was
78%.
4. Determine the quenching conditions that mediate digestion
and denaturation (see Note 4). The quenching conditions used
in the Akt experiment were 20 μL of ice cold 2 M guanidine
HCl, 3% formic acid, giving a final concentration of 0.6 M
GdHCl and 0.8% formic acid.
5. Determine which D 2 O exposure time points you want to
assess. Common time points used are 3 s, 30 s, 300 s, and
3000 s exposures, which enable probing a range of amide
hydrogen exchange rates. If this range is not sufficient to fully
sample the spectrum of amide exchange rates, different temperatures and pH values can be used [35].
3.1.2 Execution of HDX
1. Thaw the appropriate amount of protein and lipids for entire
experiment. Once thawed, keep protein on ice and lipids at
20
C.
2. Mix protein well and spin down in a cooled tabletop centrifuge
(4
C) at 15,000 rpm for 5 min to remove any aggregate. Make
sure to check the protein concentrations prior to any experimentation as the protein may not be at the same concentration
that it was originally stored at. Adjust calculations as necessary
to ensure final protein concentrations are correct.
3. Label and organize 0.6 mL Eppendorf tubes for all samples
(indicate condition and time point on label). It is ideal to have a
triplicate set of all exchange reactions. Make sure to include
tubes for at least two non-deuterated apo samples. One will be
used to identify the peptides generated during proteolysis (see
Subheading 3.3.1); the other will be used as a non-deuterated
reference that the analytic software will compare the deuterated
samples to.
4. Combine both protein and lipid components in a final volume
of 10 μL and incubate at 20
C for 2 min. For the apo samples
472
Jordan T. B. Stariha et al.