Rapiflex maldi tissue typer mass spectrometer

All analyses were performed using a rapifleXTM MALDI TissuetyperTM mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam™ 3D laser (Bruker Daltonik GmbH, Bremen, Germany). External calibration was performed using red phosphorus clusters in the m/z range of 0 to 2000. Mass measurements in negative ion reflectron mode were acquired in the m/z range of 500 to 1000. Two hundred shots were accumulated for each spectrum and the matrix suppression deflection was set to m/z 400. The samples were rastered at a lateral resolution of 20 × 20 (x,y) µm with a laser scan range of 16 µm per pixel.
For in situ MALDI-MS/MS, a single precursor ion was selected by using the smallest precursor ion selector (PCIS) window possible and dissociated using LID-LIFT™ technology, with the laser energy being set within a range of 40–70%. This process was performed until an MS/MS spectrum was obtained from the accumulation of ~100,000 laser pulses.

All imaging analyses were performed using a rapifleXTM MALDI Tissuetyper^TM mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam™ 3D laser. External calibration was performed using red phosphorus clusters in the m/z range of 0 to 2000 [26 (link)]. Mass measurements were performed in negative ion reflectron mode in the m/z range of 500 to 900. The matrix suppression deflection was set to m/z 400 and the spectrometer voltages were set as following; Ion Source 1 = 19.93 kV, PIE = 2.58 kV, Lens = 10.95 kV, Reflector 1 = 20.87 kV, Reflector 2 = 0.97 kV, Reflector 3 = 8.76 kV.
For the MALDI-MS images acquired from the thymus and tonsil, 500 shots were accumulated for each spectrum and the measurement regions were rasterised at a lateral resolution 50 × 50 (x, y) µm with a laser scan range of 44 µm per pixel. For those acquired from the colorectal cancer tissue, 200 shots were accumulated for each spectrum and the measurement regions were rasterised at a lateral resolution 10 × 10 (x, y) µm with a laser scan range of 6 µm per pixel. Laser focus adjustment was performed for each line of the tissue microarray.

Lipidomic analysis was performed as previously described [63 (link)]. In details, lipids were isolated from HCC cells JHH7 transduced with shCtl or shTG2 using the chloro-form/methanol/water extraction method. The chloroform phase containing lipids was evaporated under vacuum in SpeedVac (CC-105, TOMY, Tokyo, Japan) and reconstituted in 50 μL isopropanol (Wako Industries). Lipids were mixed with 15 mg/mL DHB matrix (1:5 v/v) in 90% acetonitrile/0.1% tri-fluoroacetic acid aqueous solution, and 0.5 μL of the mixture was loaded onto a MAL-DI-TOF target plate (MTP 384 target plate ground steel, Bruker Daltonics). Mass spectrometric analysis was performed using a rapifleX MALDI Tissuetyper mass spectrometer (Bruker Daltonics) at a spatial resolution of 20 µm. Peak calibration was carried out with a mixture of 10 mg/mL DCTB and 1 mg/mL cesium triiodide (1:1 v/v). Each collected spectrum was the sum of 10 single spectra obtained by shooting the laser at random positions on the target spot. Data were analyzed using FlexImaging software (Bruker Daltonics). Peak intensity was normalized to the protein concentration.