Determine the viral titer of the amplified viruses for each of the
AMPK subunits.- Any buffer that is free from high concentrations of salts
(>500 mM) and ionic detergents, which are not MS friendly
in later steps of the protocol, will work. We have had success
with HEPES, TRIS, and phosphate buffer systems. (Note: Tris
buffer changes pH with temperature, and it is important to use
cold solvents when making Tris buffers for HDX.) - Utmost care should be taken to minimize deuterium back
exchange (D/H) reaction with chromatography solvents. All
HPLC buffers are maintained in acidic (~pH 2.5) and refriger-
ated conditions, and short gradients are employed (t1/2of
D/H is ~30–45 min at pH 2.5 and 0C). - MS/MS sequence coverage is a function of protein concentra-
tion and is further complicated by short protease digestion
times when studying large protein systems like AMPK. Higher
protein concentration and alternative quench conditions are
often required to study large protein complexes. - While this could be done manually, use of a robot such as the
Twin PAL liquid handling robot minimizes pipetting errors
and improves data reproducibility. - The pepsin column is housed inside a temperature controlled
box held at 15C to improve digestion efficiency while sacrifi-
cing minimally on back exchange using short digestion times
(~1 min). - Immobilized protease columns provide significant advantages
over pepsin solutions in terms of digestion reproducibility and
efficiency. - Mascot is a software search engine used to identify proteins and
peptides based on mass spectrometric data. It uses a probabi-
listic scoring algorithm to identify peptides. While the software
is freely available to use on the website of Matrix Science, a
license will be required for in-house use. - Stringent quality threshold criteria should be applied to
MS/MS spectra (e.g., Mascot score, manual inspection, and
pepsin preferred and forbidden cleavage rule [21] for accurate
peptide identification). - Other denaturants such as guanidine hydrochloride and use of
strong reducing agents like TCEP have proven to be effective
for the analysis of large proteins like antibodies and membrane
proteins that contain multiple disulfide bonds. - HDX data reproducibility is affected by batch to batch varia-
bility in AMPK protein preparations. Comparison of D 2 O
exchange kinetics of Apo AMPK across various batches is
recommended for testing sample heterogeneity. Avoid
50 Ravi G. Kurumbail et al.