Synapt g2 si qtof instrument

For experiments using linear TWIM, the method outlined
above was performed but with a SYNAPT G2-Si QTOF instrument (Waters
Corporation, Wilmslow). Identical UPLC equipment, buffers, samples,
and columns were used to maximize comparability. Further details regarding
instrument tuning and mobility parameters can be found in the Supporting Information.

An intact protein MS analysis was carried out following a HILIC-based separation on a glycoprotein column. The chromatographic separation of the glycoforms was performed using a Glycoprotein BEH Amide column (Waters (Milford, MA); 300 Å, 1.7 μm, 2.1 mm × 150 mm) on a Waters Acquity UPLC system. The separation of the bioconjugate was achieved with a gradient starting at 25% of 0.1% trifluoroacetic acid (TFA) in water (buffer A) and 75% of 0.1% TFA in in acetonitrile (buffer B) going to 55% buffer A and 45% buffer B in 30 min at a flow rate of 0.2 mL/min. Detection was performed using an UV detector at 215/280. The UPLC was directly connected to a Waters Synapt G2Si Q-TOF instrument for mass determination run in resolution mode. Masses were acquired in the positive ion mode between m/z 500 and 4500 with conditions adapted for intact protein analysis. Lock mass correction was applied. MS spectra were deconvoluted with the MassLynx MaxEnt 1 algorithm (Waters) to determine the average intact masses of the glycoproteins injected. For the deconvolution, spectra belonging to an individual peak were combined, smoothed and background subtracted and the deconvolution carried out individually for each of these peaks.

MALDI-MSI was carried out using a Synapt G2-Si QToF instrument (Waters) fitted with a uMALDI ion source^{57 (link)}. The samples were coated with 9-AA (Merck Life Science) at 10 mg ml⁻¹ in 80:20 ethanol:water by TM Sprayer (HTX Technologies) with a temperature of 65 °C, flow rate of 0.06 ml min⁻¹, nozzle velocity of 1,200 mm s⁻¹, four passes, 3 ml track spacing and CC pattern alternation. The instrument was calibrated with Red Phosphorus to an RMS mass accuracy of <1 ppm, and the instrument method was set as follows: pixel size of 45 × 45 µm, sensitivity mode, negative ion mode, m/z range of 50–1,200, laser repetition rate of 2,500 Hz and scan time of 0.5 s.
DESI and MALDI-MSI data obtained from APC and APC KRAS tumours were also comprehensively evaluated for potential ion signal corresponding to SAM, SAH, homocysteine, cystathionine and methionine. On-tissue ion signal of sufficient signal-to-noise or accurate mass error was not identified for these ions. Metabolite standards were readily detected by DESI (Supplementary Table 7) but the lack of detection of endogenous ions in tissues derived from genetically engineered mouse models suggests these are below the limit of detection for the systems used in this study.