LC-MS for HX Measurements

In both HX protocols in Fig. 1, the same experimental
setup was used for mass spectrometry analysis. For whole
molecule HX analysis, after quenching of H/D exchange
with 50 mM citric buffer (pH 2.5, containing 6 M
guanidine HCl), LDH sample was immediately loaded into
a sample loop. An isocratic pump delivered the LDH
sample in the loop to a peptide trapping column (1 mm
ID×8 mm, catalog No. TR1/25108/01; Michrom Bioresources,
Auburn, CA) for desalting. After 6 min desalting,
a short gradient of acetonitrile (ACN) from 25% to 90%
over 7 min (50 μL/min by Surveyor MS Pump, Thermo,
San Jose, CA) was used to elute LDH from the trapping
column and deliver it to the electrospray ionization ion-trap
mass spectrometer (LTQ, Thermo Electron Corporation,
San Jose, CA).
In the peptide level HX analysis, an in-line proteolytic
digestion was incorporated by including an immobilized
pepsin column to the LC setup before the peptide trapping
column for desalting. The high concentration of guanidine
HCl introduced in the H/D quenching step would be
detrimental to the pepsin activity. Therefore, after quenching,
five-fold dilution of LDH sample into 0.1% formic acid (pH
2.5) was performed. The peptide mixture resulting from
pepsin column digestion was desalted with the peptide
trapping column described above, and then separated in a
second peptide-resolving column (Kinetex 2.6 μmC18, 2.10×
100 mm, Phenomenex, Torrance, CA). For good peptide
separation, a shallower ACN elution gradient (from 15 to
40% over 20 min) was used. All the reporter peptides used
for HX analysis were assigned by performing tandem (MS/
MS) mass spectrometry, followed by analysis with TurboSEQUEST
software. To minimize artifactual isotope exchange
during the analysis time, all the columns, loops, and lines
were immersed in an ice bath during all the experiments.
The deuteration level for intact molecule and each
reporter peptide was calculated by the following equation:
D% ¼ m m0
ðm100 m0Þ
100% ð1Þ
where m is the measured centroid mass of the deuterated
molecule or peptide after a particular labeling time, and m0
and m100 are the two centroid mass limits of a molecule and
reporter peptide from zero-deuteration and full-deuteration
control experiments, respectively.