Manual icebox LC MS protocol for HDX samples

Operating the LC/MS and collection of MS data

1. Check that VICI box is set to A.
2. Check 0.1% formic acid solution is full – if not, make some more.
3. Loosen the 2 black valves on HPLC and place beakers under them, then press the buttons to turn the pumps on.
4. Switch on the software.
5. Purge the top (CAP) pump lines: prime flow, 2500/5 min, channels A1 and B1 (water + acetonitrile). For the bottom (QUAT) pump – switch it on and set the method to flow at 5 ml/min (water only). No purge option on bottom pump. Let the lines wash until the CAP pump is done purging.
6. Switch pumps off by setting the method to flow rate = 0 and retighten the black valves on the HPLC.
7. Attach the trap and pepsin columns to the lines where labeled – either direction fine.
8. Turn on bottom pump to flow at 0.4 ml/min. The pressure should be ~25-40 bars.
9. Set the top pump to flow at 12 µl/min normal flow. Add the analytical column such that the arrow is facing the incoming line, with flow into the guard column first.
10. Then immediately switch to micro flow with 17 µl/min.
11. Check for waste coming out of the red line on the cart and the black line under the cart. The pressure should be around ~140 bars.
12. Standard flow through top pump: 85% water, 15% acetonitrile.
13. Move the black handle in the cooler to “LOAD”.
14. Check for liquid on the nozzle in the MS.
15. Load (~17-min) method and hit the yellow button on the left in the MS software to start the MS.
16. Check for 4 kV and the sheath gas flow rate at 30.
17. Wash the syringe out by sucking 0.05% TFA solution into it a few times, load sample and eject bubbles, then inject into the line (do a blank first – just 0.05% TFA solution).
18. Make a file (blank) and find methods (XCaliber HDX/MS folder).
19. Set to contact closure so that the method starts after you manually move the handle to “INJECT” and also select “go to standby when finished”.
20. Press START. It will say “waiting to start”.
21. Make sure you injected the sample into the line.
22. Switch to “INJECT” by moving the handle clockwise all the way.
23. The green IDLE bar on the HPLC will change to blue and say RUNNING, and the MS window will change the state to “acquiring”.
24. It's now cool to open HDExaminer and PeptideExplorer on the analysis computer.
25. Turn handle from “INJECT” back to “LOAD” after ~20 min collecting data.
26. Wait for the pressure to be constant in both pumps, then run the MS/MS sample the same way after a few minutes, except with the MS-MS protocol instead of HDX-MS.
27. If there are a good number of peptides (>75) and PSMs (~300-400) from MS/MS according to PeptideExplorer, fill the cooler with ice water so that all the lines are submerged, switch the method back to HDX-MS, and run the samples, starting with a water sample for the zero TP.

Shut down protocol

1. 15-20 minute wash in the top pump of acetonitrile
2. Switch off and wait for both pumps to stop
3. Put away columns
4. Turn off the four buttons on the HPLC
5. Turn off the switch on the outlet hanging from the ceiling
6. Export data