Hydrogen deuterium exchange mass spectrometry (HDX-MS)

HDX-MS reveals nucleotide-dependent, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)

Protocol:

1. Mix purified SecYEG and SecA at 15 μM and 10 μM, respectively, to a total volume of 100 μL for experiments investigating SecA. Or, mix purified SecYEG and SecA at 10 μM and 15 μM, respectively, to a total volume of 100 μL for experiments investigating SecYEG. 
2. For experiments analysing the Sec complex in the presence of nucleotides, a 50mM stock of AMPPNP or ADP was added to the SecYEG-SecA complex to a final volume of 1mM.
3. Incubate the SecYEG-SecA complex for 10 minutes on ice
4. For experiments analysing the Sec complex in the presence of nucleotides, add a 50mM stock of AMPPNP or ADP in D₂O to the deuteration buffer to a final volume of 1mM.
5. Add 5 μL SecYEG-SecA complex to 95 μL deuteration buffer or equilibration buffer. 
6. At 25°C, incubate the SecYEG-SecA complex in deuteration buffer for 0.25, 1, 5, or 30 minutes
7. Quench the deuteration by adding 100 μL quench buffer pre-chilled to 1°C
8. The following steps were carried out after individually injecting reaction mixtures onto a Synapt G2-Si HDMS coupled to an Acquity UPLC M-Class system with HDX and automation
9. Digest quenched reaction mixtures on an Enzymate online digestion column (Waters) in 0.1% (v/v) formic acid at a flow rate of 200 μL/min at 25°C. In between injection of samples, wash the pepsin column with cleaning solution. Furthermore, run a blank sample (e.g. solution of 0.8% (v/v) formic acid) between sample runs
10. Trap peptides with an Acquity BEH C18 1.7 μM VANGUARD pre-column for 3 min at a flow rate of 200 μL/min in buffer A (0.1% (v/v) formic acid at a pH of ~2.5)
11. Elute peptides into an Acquity UPLC BEH C18 1.7 μM 1.0 × 100 mm analytical column with a linear gradient of 8–40% (v/v) gradient of acetonitrile with 0.1% (v/v) formic acid at a flow rate of 40 μL/min
12. Analyse peptides in a Synapt G2-Si mass spectrometer (Waters) using positive ion mode in the electrospray ionisation source. Enable ion mobility for all experiments. Use a 20–30 V trap collision energy ramp to capture the MS^E data. Use Leucine Enkephalin as a lock mass and iodide for mass spectrometry calibration.
13. Repeat all deuterated time points in triplicate

Solutions:

1. Deuteration buffer: 20 mM Tris pH 8, 2 mM MgCl₂, 50 mM KCl, and 0.02% (w/v) dodecyl-maltoside (DDM) in D₂O
2. Equilibration buffer: 20 mM Tris pH 8, 2 mM MgCl₂, 50 mM KCl, and 0.02% (w/v) DDM in H₂O
3. Quench buffer: 0.7% (v/v) formic acid and 0.1% (w/v) DDM
4. Cleaning solution: 0.8% (v/v) formic acid, 1.5 M Gu-HCl, and 4% (v/v) MeOH

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