Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

Complexes were formed on ice and incubated for 30 mins giving a final Parkin concentration of 10 μM. Deuterium exchange reactions of Parkin and the different complexes were initiated by diluting the protein in D₂O (99.8% (v/v) D₂O ACROS, Sigma, UK) in 25 mM Tris (pH 8.5), 200 mM NaCl, 1 mM TCEP to give a final D₂O percentage of ~95%. For all experiments, deuterium labelling was carried out at 23°C (unless otherwise stated) at five time points, 0.3 seconds (3 seconds on ice), 3 seconds, 30 seconds, 300 seconds and 3000 seconds, in technical triplicate. The labelling reaction was quenched by the addition of chilled 2.4% (v/v) formic acid in 2 M guanidinium hydrochloride and immediately frozen in liquid nitrogen. Samples were stored at -80°C prior to analysis.

The quenched protein samples were rapidly thawed and subjected to proteolytic cleavage with pepsin followed by reversed phase HPLC separation. Briefly, the protein was passed through an Enzymate BEH immobilised pepsin column, 2.1 x 30 mm, 5 μm (Waters, UK) at 200 μL/min for 2 min, the peptic peptides were trapped and desalted on a 2.1 x 5 mm C18 trap column (Acquity BEH C18 Van-guard pre-column, 1.7 μm, Waters, UK). Trapped peptides were subsequently eluted over 11 min using a 3-43% gradient of acetonitrile in 0.1% (v/v) formic acid at 40 μL/min. Peptides were separated on a reverse phase column (Acquity UPLC BEH C18 column 1.7 μm, 100 mm x 1 mm (Waters, UK) and detected on a SYNAPT G2-Si HDMS mass spectrometer (Waters, UK) over an m/z of 300 to 2000, with the standard electrospray ionisation (ESI) source with lock mass calibration using [Glu1]-fibrino peptide B (50 fmol/μL). The mass spectrometer was operated at a source temperature of 80°C and a spray voltage of 2.6 kV. Spectra were collected in positive ion mode.

Peptide identification was performed by MS^{e 36} using an identical gradient of increasing acetonitrile in 0.1% (v/v) formic acid over 11 min. The resulting MS^e data were analysed using Protein Lynx Global Server software (Waters, UK) with an MS tolerance of 5 ppm.

Mass analysis of the peptide centroids was performed using DynamX software (Waters, UK). Only peptides with a score >6.4 were considered. The first round of analysis and identification was performed automatically by the DynamX software, however, all peptides (deuterated and non-deuterated) were manually verified at every time point for the correct charge state, presence of overlapping peptides, and correct retention time. Deuterium incorporation was not corrected for back-exchange and represents relative, rather than absolute changes in deuterium levels. Changes in H/D amide exchange in any peptide may be due to a single amide or a number of amides within that peptide.