Synapt g2 s hdms mass spectrometer

B. subtilis 168/DSM402 was grown in chemically defined Belitzky minimal medium (BMM)61 (link). Cultures were incubated at 37 °C under steady agitation. Minimal inhibitory concentrations (MIC) were determined as described previously62 (link). In growth experiments, exponentially growing cultures were exposed to a concentration range of peptide (Supplementary Figure 1)62 (link). For physiological stress experiments, concentrations were chosen that led to 50% reduction of the growth rate. For proteomic profiling, as well as for all follow up experiments performed under the same growth conditions, 8 μM cWFW were used.
Radioactive labelling of newly synthesised proteins and subsequent separation of the cytosolic proteome by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed as previously described62 (link). Analytical gel images were analysed as described by Raatschen & Bandow63 using Decodon Delta 2D 4.1 image analysis software. Proteins more than two-fold upregulated in three independent biological replicates were defined as marker proteins. Protein spots were identified by MALDI-ToF/ToF or nanoUPLC-ESI-MS/MS using a Synapt G2-S HDMS mass spectrometer equipped with a lock spray source for electrospray ionisation and a ToF detector (Waters) as previously reported (Supplementary Tables 2 and 3)62 (link).

High resolution mass spectra were recorded on a Waters Synapt G2-S HDMS mass spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA, USA) consisting of a binary solvent manager, sample manager, and column oven. For screening of extracts, aliquots (5 µL) of all samples were injected into the UPLC-TOF-MS system using a BEH C18 column (150 mm × 2.1 mm, 1.7 µm, Waters), operated at a flow rate of 0.4 mL/min at 45 °C. Chromatographic separation was done with a gradient of aqueous formic acid (0.1%, A) and acetonitrile (0.1%, B). The gradient started with a mixture of 1% B and was kept isocratic for 1 min, then, increased to 100 % B within 9 min and held at 100 % B for 1 additional min. Measurements were performed in high resolution mode with negative electrospray ionization (ESI−) and positive electrospray ionization (ESI+). The ion source parameters were as follows: capillary voltage +2.5 kV (ESI+) or -2.5 kV (ESI−), sampling cone 50 V, source offset 30 V, source temperature 150 °C, desolvation temperature 450 °C, cone gas 2 L/h, nebulizer gas flow 6.5 bar, and desolvation gas 850 L/h. Data processing was handled with MassLynx 4.1 (Waters) and Progenisis QI (Waters).

LC-MS/MS was used to identify and quantify DISC proteins (see Supplemental Experimental Procedures). Briefly, TRAIL and CD95 DISC proteins were separated by SDS-PAGE and serial gel slices digested in situ with trypsin (Dickens et al., 2012a (link)). Extracted tryptic peptides were analyzed using data-independent acquisition (DIA) on a nanoAcquity UPLC system coupled to a Waters Synapt G2-S HDMS mass spectrometer. The PLGS “TOP 3” method was used for absolute quantification of proteins (Silva et al., 2006 (link)).