- Initiate each exchange reaction by incubating 4μl of AMPK
protein (with or without ligand) with 16μl of AMPK D 2 O
buffer for a predetermined time (10 s, 30 s, 60 s, 300 s, 900 s,
and 3600 s in a randomized order) on a 96-well plate setup.
Sample and D 2 O buffer volume can be adjusted using concen-
trated protein samples, and incubation times can be varied to
get maximum deuterium incorporation (seeNotes 26–29). - Slow the exchange reaction rate by mixing the protein solution
with 30μl of ice-cold quench solution (seeNote 16). - For peptide identification, collect three replicates of control
undeuterated AMPK sample mixed with H 2 O HDX buffer for
time 0 s and quenched similar to D 2 O-containing samples. - Pass the quenched reaction mixture (50μl) through the immo-
bilized pepsin column at 50μl/min, and capture the digested
peptides onto a C8 trap column and desalt (total time for
digestion and desalting is 2.5 min). - Separate the peptides using a C18 analytical column with a
short 8-min linear gradient (5–40% ACN and 0.3% formic acid
over 5 min, then increased to 100% ACN for 2 min, and finally
reduced to 5% ACN over 1 min). - Protein digestion and peptide separation steps utilized for
HDX-MS experiments are identical to MS/MS except for the
use of a time-compressed gradient used in HDX-MS runs to
reduce D/H back exchange with chromatography solvents and
preserve exchanged deuterium (seeNote 16). - Electrospray ionization and MS data acquisition parameters are
set similar to the tandem MS experiments described above. - Perform three technical replicates for each on-exchange time
point to allow statistical analysis of the data.
For HDX data analysis, followsteps 30– 34 below.- Load data from mass spectrometer into HDX Workbench [29]
(previous versions include Deuterator [30] and HD Desktop
[31]) or similar software. Other software packages can be used,
but they should be capable of handling high-resolution
HDX-MS data that are often large and should also support
ligand comparisons (seeNote 30). - Check that sufficient sequence coverage and unambiguous
peptide identification (mass accuracy<3 ppm for precursor
monoisotopic mass) are obtained in the initial peptide detec-
tion step using undeuterated control samples before proceed-
ing to estimate percent deuterium incorporation. Sequence
coverage on HDX datasets is comparatively lower than the
MS/MS run due to peptide loss in shorter HDX gradient,
ion suppression by complex samples, and multiple injections
40 Ravi G. Kurumbail et al.