irreproducible results. Buffer pH should be adjusted and ver-
ified using standard commercial pH meters and also double-
checked with pH strips for accuracy.
- Correct for pH of D 2 O buffers by adjusting the actual reading
from the pH meter to take in to account electrode differences
between reading protons and deuterons. pD¼actual pH meter
readingþ0.4.
- Confirm that pH of quenched samples is 2.5 or below after
mixing with protein sample buffer. These pH tests can be made
in solutions of the buffer that do not contain protein.
- Store quench solution at 4C while in use (~1 week) or at
80 C for long-term storage (>1 week).
- Modern-day mass spectrometers capable of high-resolution
data acquisition (minimum resolving power of 60,000 at m/z
400) are needed for accurately assigning peptide peaks and
reliable percent deuterium calculations for high MW
(>80 kDa) proteins and protein complexes. An Exactive Orbi-
trap instrument capable of a resolving power of 100,000 at
m/z 400 was used for the HDX experiments reported in Land-
graf et al. [11].
- A typical setup for running of HDX experiments requires
HPLC columns, robotics, and protease columns integrated
with a high-resolution mass spectrometer. We have described
the instrumentation setup in our lab, but other combinations
of the individual components are also possible.
- It is critical that the amount of DMSO is matched between
samples and running buffer (Buffer B). Making up the samples
using the original buffer solutions will help to eliminate refrac-
tive index jumps that could otherwise be observed if the buffer
and samples are not matched.
- When making Buffers A and B, it is important to use degassed
water to help eliminate bubbles in the samples and the possi-
bility of injecting air.
- Although the pET14b expression vector contains built-in
DNA for a His-tag and a thrombin cleavage site, we
incorporated the coding sequence for a His-tag into our syn-
thetic tricistronic expression plasmid for increased flexibility in
positioning the His-tag at the appropriate location of AMPK
subunits. Cleaving the vector withNcoIandXhoIrestriction
enzymes removes the His-tag and thrombin site from the
vector.
- Generate P0 viruses containing individual recombinant AMPK
α,β, andγsubunits. Amplify the viruses by infecting Sf-9 cells
with P0 viruses and thus generate P1, P2, etc., viruses.
Biophysical Studies to Evaluate Protein-Ligand Interactions 49
