18. Dry sample in a vacuum dryer.
19. Redissolve in 20 μL of 1% FA. Sonicate 5 min in an ultrasonic
bath and centrifuge at maximum speed (16,000 Â g) for 5 min.
20. Transfer carefully the protein digest to an autosampler vial
(with insert) (see Note 69).
3.6 LC–MS/MS
Analysis
of Carbonylome
1. Before starting, ensure that there is enough volume in solvent
reservoirs. Changed/refilled if necessary.
2. If the solvent reservoirs have been changed/refilled, the system
needs to be purged. To purge the loading pump (Micro pump),
just open the corresponding valve by rotating counterclockwise. Click the button “purge on” from the pump main window. After 12 min, when the purge automatically finishes, close
the valve by rotating clockwise. Slowly restore the flow up to
reach 10 μL/min.
3. Regarding the purge of the Nano pump, select the first purge
“both blocks” in the pump window. Open the valves on each
block by rotating counterclockwise and click the button “purge
on.” Both block purge last 30 min. When finish, close the
valves by rotating clockwise and disconnect the peak column
from the outlet. Select now purge “flowmeter” in the pump
main window and click the button “purge on.” Flowmeter
purge last 30 min. Perform afterward pressure and viscosity
tests by following the instructions. Accept the new viscosity
values for the mobile phases if they have changed. If everything
is correct, connect the column peak to the outlet of the Nano
pump. Slowly restore the flow up to reach 300 nL/min and
initial conditions (95% mobile phase A, 5% mobile phase B).
Maintain conditions to equilibrate the column (see Note 70).
4. Check the tune of the mass spectrometer (see Note 71).
5. Inject 3 μL of the digest of carbonylated protein from each of
detected spots onto the trap column at a flow rate of 10 μL/
min by loading pump for 3 min (see Note 72).
6. Load the desalted peptide mixture to a C18 column at a
constant flow rate of 300 nL/min by Nano pump (95% mobile
phase A, 5% mobile phase B) to perform the peptide separation.
7. Separate the peptides using a linearly increasing concentration
of AcN in buffer B given in Table 1.
8. Perform tandem mass spectrometry (MS/MS) analysis with
LIT mass spectrometer operated in data-dependent acquisition
(DDA) mode. Acquire MS1 survey scans in the mass range of
mass/charge ratio (m/z) 400 to 1600 Da. Select the six mostintense precursor ions with !2 charge state and fragment in the
ion trap by collision-induced dissociation (CID) with 35%
Fluorescent 2DE-LC-MS for Carbonylome Analysis
239
19. Redissolve in 20 μL of 1% FA. Sonicate 5 min in an ultrasonic
bath and centrifuge at maximum speed (16,000 Â g) for 5 min.
20. Transfer carefully the protein digest to an autosampler vial
(with insert) (see Note 69).
3.6 LC–MS/MS
Analysis
of Carbonylome
1. Before starting, ensure that there is enough volume in solvent
reservoirs. Changed/refilled if necessary.
2. If the solvent reservoirs have been changed/refilled, the system
needs to be purged. To purge the loading pump (Micro pump),
just open the corresponding valve by rotating counterclockwise. Click the button “purge on” from the pump main window. After 12 min, when the purge automatically finishes, close
the valve by rotating clockwise. Slowly restore the flow up to
reach 10 μL/min.
3. Regarding the purge of the Nano pump, select the first purge
“both blocks” in the pump window. Open the valves on each
block by rotating counterclockwise and click the button “purge
on.” Both block purge last 30 min. When finish, close the
valves by rotating clockwise and disconnect the peak column
from the outlet. Select now purge “flowmeter” in the pump
main window and click the button “purge on.” Flowmeter
purge last 30 min. Perform afterward pressure and viscosity
tests by following the instructions. Accept the new viscosity
values for the mobile phases if they have changed. If everything
is correct, connect the column peak to the outlet of the Nano
pump. Slowly restore the flow up to reach 300 nL/min and
initial conditions (95% mobile phase A, 5% mobile phase B).
Maintain conditions to equilibrate the column (see Note 70).
4. Check the tune of the mass spectrometer (see Note 71).
5. Inject 3 μL of the digest of carbonylated protein from each of
detected spots onto the trap column at a flow rate of 10 μL/
min by loading pump for 3 min (see Note 72).
6. Load the desalted peptide mixture to a C18 column at a
constant flow rate of 300 nL/min by Nano pump (95% mobile
phase A, 5% mobile phase B) to perform the peptide separation.
7. Separate the peptides using a linearly increasing concentration
of AcN in buffer B given in Table 1.
8. Perform tandem mass spectrometry (MS/MS) analysis with
LIT mass spectrometer operated in data-dependent acquisition
(DDA) mode. Acquire MS1 survey scans in the mass range of
mass/charge ratio (m/z) 400 to 1600 Da. Select the six mostintense precursor ions with !2 charge state and fragment in the
ion trap by collision-induced dissociation (CID) with 35%
Fluorescent 2DE-LC-MS for Carbonylome Analysis
239