lipid phosphoinositides and a kinase domain. When the PH domain
is not bound to phosphoinositides, Akt is soluble and unphosphorylated at two key phosphorylation sites critical for its activity,
T308 and S473. This renders soluble Akt in an autoinhibited,
inactive state where the PH domain is engaged in a direct interaction with the kinase domain. When Akt is recruited to membranes
containing PIP 3 or PI3,4P 2 , the PH domain inhibitory interface is
disrupted, and it can be phosphorylated at T308 and S473, leading
to full activation. The HDX-MS experiments described below
investigated the differences in H–D exchange for Akt in the presence and absence of 5% PIP 3 membranes to identify conformational
changes that mediate the transition between the autoinhibited and
activated form.
2 Materials
2.1 Instrumentation
Buffers
Important note: All buffers used on the mass spectrometer
(MS) should be detergent-free. Use new glassware for making and
storing all buffers that are used for the upkeep of the MS and
sample preparation to avoid residual detergent. The easiest way to
do this is buy ultra-grade reagents, and use in the bottles they are
sold in.
1. Digestion buffer: 0.05% LC/MS grade trifluoroacetic acid
(TFA; pH 2.5). Sterile filter 2 L of deionized water into a
detergent-free bottle using a 0.22 μm filter. Add 1 mL 100%
LC/MS TFA into the 2 L of filtered deionized water to give a
final concentration of 0.05% TFA. Pour off a small volume of
the final buffer into a separate container and measure the pH to
confirm it is at the desired value of 2.5.
2. Needle wash 1: 0.05% LC/MS grade trifluoroacetic acid (TFA;
pH 2.5). Use the same protocol as the Digestion Buffer
detailed above for making this buffer.
3. Needle wash 2: 10:90 HPLC grade acetonitrile (ACN):
H 2 O + LC/MS grade 0.1% formic acid (FA). Combine
100 mL of ACN with 900 mL of 0.1% FA and filter the mixture
through a 0.22 μm filter unit.
4. Sample loop/pepsin wash: 0.1% LC/MS grade formic acid
(FA) + 1 M guanidine. Measure out 4.78 g of guanidine
hydrochloride into a new sterile 50 mL falcon. Add LC/MS
grade 0.1% FA up to the 50 mL mark, dissolve the guanidine by
vortexing and/or sonication, and filter the mixture through a
0.22 μm filter. Store at À20
C.
5. Mobile phase A: 0.1% LC/MS grade formic acid (FA).
6. Mobile phase B: 100% LC/MS grade acetonitrile (ACN).
Probing Protein-Membrane Interactions Using HDX-MS
467
is not bound to phosphoinositides, Akt is soluble and unphosphorylated at two key phosphorylation sites critical for its activity,
T308 and S473. This renders soluble Akt in an autoinhibited,
inactive state where the PH domain is engaged in a direct interaction with the kinase domain. When Akt is recruited to membranes
containing PIP 3 or PI3,4P 2 , the PH domain inhibitory interface is
disrupted, and it can be phosphorylated at T308 and S473, leading
to full activation. The HDX-MS experiments described below
investigated the differences in H–D exchange for Akt in the presence and absence of 5% PIP 3 membranes to identify conformational
changes that mediate the transition between the autoinhibited and
activated form.
2 Materials
2.1 Instrumentation
Buffers
Important note: All buffers used on the mass spectrometer
(MS) should be detergent-free. Use new glassware for making and
storing all buffers that are used for the upkeep of the MS and
sample preparation to avoid residual detergent. The easiest way to
do this is buy ultra-grade reagents, and use in the bottles they are
sold in.
1. Digestion buffer: 0.05% LC/MS grade trifluoroacetic acid
(TFA; pH 2.5). Sterile filter 2 L of deionized water into a
detergent-free bottle using a 0.22 μm filter. Add 1 mL 100%
LC/MS TFA into the 2 L of filtered deionized water to give a
final concentration of 0.05% TFA. Pour off a small volume of
the final buffer into a separate container and measure the pH to
confirm it is at the desired value of 2.5.
2. Needle wash 1: 0.05% LC/MS grade trifluoroacetic acid (TFA;
pH 2.5). Use the same protocol as the Digestion Buffer
detailed above for making this buffer.
3. Needle wash 2: 10:90 HPLC grade acetonitrile (ACN):
H 2 O + LC/MS grade 0.1% formic acid (FA). Combine
100 mL of ACN with 900 mL of 0.1% FA and filter the mixture
through a 0.22 μm filter unit.
4. Sample loop/pepsin wash: 0.1% LC/MS grade formic acid
(FA) + 1 M guanidine. Measure out 4.78 g of guanidine
hydrochloride into a new sterile 50 mL falcon. Add LC/MS
grade 0.1% FA up to the 50 mL mark, dissolve the guanidine by
vortexing and/or sonication, and filter the mixture through a
0.22 μm filter. Store at À20
C.
5. Mobile phase A: 0.1% LC/MS grade formic acid (FA).
6. Mobile phase B: 100% LC/MS grade acetonitrile (ACN).
Probing Protein-Membrane Interactions Using HDX-MS
467