Smartbeam 3d laser

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.

MALDI-TOF-MS in reflection positive-ion mode was used for the serum N-glycans profiling (17 (link)). 1 μL of glycan samples was mixed with 1 μL of matrix containing 5 mg/mL super-DHB with 1 mM NaOH in 50% ACN on the AnchorChip target plate (Bruker Daltonics, Bremen, Germany) and the mixture was dried by air for 2 h. The N-glycomes were detected using a rapifleXtreme MALDI-TOF mass spectrometer with a Smartbeam-3D laser, controlled by flexControl 4.0 (Bruker Daltonics). A peptide calibration standard (Bruker Daltonics) was used for the calibration of the instrument. The mass range was set from m/z 1000 to m/z 5000. Laser shots were accumulated 5000 times for each spectrum. We chose an automatic acquisition mode and random walk pattern at a laser frequency of 5000 Hz for the acquisition of sample spectra (17 (link)).

MALDI MSI data were acquired with a rapifleX tissuetyper in single TOF mode (Bruker Daltonik GmBH, Bremen, Germany), equipped with a SmartBeam 3D laser. Mass spectra were the sum of 1000 individual laser shots, with a 90% laser intensity. Mass spectral peptidomic (m/z range 800 Da–5 kDa) images were obtained in positive reflector mode with a reflector voltage of 3005 V, a sample rate of 0.63 GS/s, a laser resolution of 50 µm and a raster width of 50 µm × 50 µm.
All the spectra are preprocessed with a Top Hat baseline algorithm for baseline subtraction and the resulting overall average spectrum of the ion image is TIC normalized in flexImaging 5.0 (Bruker Daltonik GmBH, Bremen, Germany) after recalibration in flexAnalysis 4.0 with external calibration standard. The results will be further processed in SCiLS lab 2016b (Bruker Daltonik GmBH, Bremen, Germany) and R software (Cardinal) [42 (link)].