LacI HDX exchange protocol

Buffers

• Quench buffer (2X): 6M urea, 200mM arginine, 100mM TCEP, pH 2.0
• Protein buffer: 50mM HEPES, 150mM NaCl, 0.5mM TCEP pH 7.5
• Exchange buffer: same as protein buffer, but lyophilized 2X overnight to remove H2O and with D2O added back to an equal volume

Check all pH values!

Protein prep

Time points: 0 s, 15 s, 30 s, 45 s, 1 min, 5 min, 15 min, 1 h, 2 h, 4 h Label tubes for all TPs.

Thaw aliquot of the protein sample at 1 mg/ml, spin 20 min at max speed at 4C in tabletop centrifuge, and filter the supe.

Exchange

1. Pipette 100 µl quench into all TP tubes.

2. Dilute sample to final concentration of ~6 µM protein solution in exchange buffer (D2O, 1:10), and start timer.

3. Immediately start taking TPs, adding 100 µl protein sample, mixing quickly with quench buffer, and freezing in liquid N2.

All samples will have ~3 µM final protein concentration in 200 µl volumes.

Don't forget to save enough protein for MS/MS on a few water samples (no exchange).

Storage

-80C

Benchtop pepsin digest, if you don't have a column, but you do have frozen soluble pepsin aliquots.

• For all steps, try to avoid touching the tube where the sample is, because if we heat up any part of the sample too much, we'll increase the back-exchange.
• Thaw the sample by dipping briefly in room temp H2O, wiping the side of the tube quickly, and then tapping until the ice starts to melt.
• Place the sample on ice.
• Thaw your 142 µM soluble pepsin aliquot.
• Once melted, quickly add 14 µl of pepsin, recap the tube, tap a couple of times to mix, and place back on ice. Have your labmate start a timer the second you add the pepsin. (Concentration should be 2 µM, volume should be 114 µl)
• After 3 min, apply ~70 µl of the sample to the LC loop.