Quench buffers

Different quench buffers work best for different proteins.

Sometimes it takes some optimization to determine which is the best quench buffer for your protein. The best approach for bottom-up HX/MS experiments is to optimize the quench buffer by running a few MS/MS experiments to see which gives you a good peptide list before setting up a full HX/MS timecourse.

Digestion conditions are intended to optimize:

• the type of denaturant
• the concentration of denaturant to get the best digestion coverage
• the type of acidic species best-suited to the protein

Proteins with disulfide bonds and membrane proteins generally require a little more fine-tuning.

This is because you want to maintain a low pH around 2.3 to minimize back-exchange. Higher pH will encourage deprotonation of TCEP, and higher TCEP concentrations (>1 M) require increased digestion/desalting time to flush all the TCEP out prior to elution. TCEP can form adducts that become problematic during the analysis, but increased chromatography timescales increase the rate of back-exchange. So you want to maintain the best pH to enhance the efficiency of TCEP, but minimize back-exchange.

Common quench buffers

Quench if not disulfide bonded and/or membrane protein	Quench if disulfide bonded and/or membrane protein
1.6 M GuHCl, 0.8% FA, pH 2.3	2 M Urea, 1 M TCEP, pH 3.0 (a)
3.2 M GuHCl, 0.8% FA, pH 2.1	8 M Urea, 1 M TCEP, pH 3.0 (a)
3.2 M GuHCl, 0.8% FA, pH 2.7 (a)	2 M Urea, 1 M TCEP, pH 4.0 (a)
2 M Urea, 1 M TCEP, pH 1.7	8 M Urea, 1 M TCEP, pH 4.0 (a)
8 M Urea, 1 M TCEP, pH 2.5	8 M Urea, 1 M TCEP, pH 4.0 (b)
8 M Urea, 1 M TCEP, pH 2.5 (c)	8 M Urea, 1 M TCEP, pH 4.0 (c)

Notes.
a) pH adjusted (down with HCl, up with NaOH).
b) pH adjusted, quench incubated at 23 °C for 3 hours.
c) 0.1% fos-choline-12 (FC12) added.

For membrane proteins, FC12 or another detergent can help with the sticky hydrophobic regions.

Additionally, you can run water blanks in between samples to identify carryover, which can be particularly bad with membrane proteins. If you observe carryover of either the protein or the quench reagents, it's best to add a column washing protocol or additional water blanks to ensure that you have a clean baseline.