Hydrogen deuterium exchange mass spectrometry (HDX-MS)

HDX experiments were performed similarly as described before (Dornan et al., 2017). For HDX with mutants, 3 μL containing 13 picomoles of protein was incubated with 8.25 μL of D₂O buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 98% (v/v) D₂O) for four different time periods (3, 30, 300, 3000 s at 20°C). After the appropriate time, the reaction was stopped with 57.5 μL of ice-cold quench buffer (2M guanidine, 3% formic acid), immediately snap frozen in liquid nitrogen and stored at −80°C. For HDX with inhibitors, 5 μL of p110γ or p110γ/p101 at 2 μM was mixed with 5 μL of inhibitor at 4 μM in 10% DMSO or 5 μL of blank solution containing 10% DMSO and incubated for 20 min on ice. A total of 40 μL of D2O buffer was added to this solution to start the exchange reaction which was allowed to proceed for four different time periods (3, 30, 300, 3000 s at 20°C). After the appropriate time, the reaction was terminated with 20 μL of ice-cold quench buffer and the samples were frozen.

Protein samples were rapidly thawed and injected onto an ultra-high-pressure liquid chromatography (UPLC) system at 2°C. Protein was run over two immobilised pepsin columns (Trajan, ProDx protease column, PDX.PP01-F32 and Applied Biosystems, Porosyme, 2-3131-00) at 10°C and 2°C at 200 μL/min for 3 min, and peptides were collected onto a VanGuard precolumn trap (Waters). The trap was subsequently eluted in line with an Acquity 1.7 μm particle, 100 × 1 mm² C18 UPLC column (Waters), using a gradient of 5–36% B (buffer A, 0.1% formic acid; buffer B, 100% acetonitrile) over 16 min. Mass spectrometry experiments were performed on an Impact II TOF (Bruker) acquiring over a mass range from 150 to 2200 m/z using an electrospray ionisation source operated at a temperature of 200°C and a spray voltage of 4.5 kV. Peptides were identified using data-dependent acquisition methods following tandem MS/MS experiments (0.5 s precursor scan from 150 to 2000 m/z; 12 0.25 s fragment scans from 150 to 2000 m/z). MS/MS datasets were analysed using PEAKS7 (PEAKS), and a false discovery rate was set at 1% using a database of purified proteins and known contaminants.

HD-Examiner software (Sierra Analytics) was used to automatically calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state and presence of overlapping peptides. Deuteration levels were calculated using the centroid of the experimental isotope clusters. The results for these proteins are presented as the raw percent deuterium incorporation, as shown in Supplemental Data, with the only correction being applied correcting for the deuterium oxide percentage of the buffer utilised in the exchange (62% for experiments with mutants and 75.5% for experiments with inhibitors). No corrections for back exchange that occurs during the quench and digest/separation were applied. Attempts to generate a fully deuterated class I PI3K sample were unsuccessful, which is common for large macromolecular complexes. Therefore, all deuterium exchange values are relative.

Changes in any peptide at any time point greater than both 5% and 0.4 Da between conditions with a paired t test value of p<0.01 were considered significant. Peptides that crossed these criteria were mapped onto the structures in Figure 2A+B and Figure 4B-D. Peptides that crossed this threshold are annotated on the raw HDX data in the source data excel file according to the legend. The raw HDX data are shown in two different formats. The raw peptide deuterium incorporation graphs for a selection of peptides with significant differences are shown, with the raw data for all analysed peptides in the source data. To allow for visualisation of differences across all peptides, we utilised number of deuteron difference (#D) plots. These plots show the total difference in deuterium incorporation over the entire H/D exchange time course, with each point indicating a single peptide. These graphs are calculated by summing the differences at every time point for each peptide and propagating the error (example Figure 2C–F). This visualisation was utilised over the similar strategy of graphing differences at each timepoint separately as it allowed for the display of multiple comparisons on the same graph (example Figure 4A).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019) with the dataset identifier PXD021132.