Scils 2016b

Released N-linked glycan ions were detected using a Solarix dual source 7T FTICR mass spectrometer (Bruker Daltonics) (m/z 490–5000) with a SmartBeam II laser operating at 2000 Hz, and a laser spot size of 15 μm. A total of 200 laser shots were collected for each pixel, using the smartwalk feature set to 25 μm with one scan per pixel. Time domain was set to 512K word with a mass range of 500–5,000 m/z, resulting in a 1.2059 transient with a calculated resolving power of 160,000 at m/z 400. Ion accumulation time was 0.1 second. Following MS analysis, data was loaded into FlexImaging Software focusing on the range m/z = 700–4000. FlexImaging 4.0 (Bruker Daltonics) was used to generate images of differentially expressed glycans normalized to total ion current. Observed glycans were searched against the glycan database generated using GlycoWorkbench [31 (link)]. Indicated glycan structures were generated in GlycoWorkbench and represent compositionally correct structures determined by accurate mass, as well as previous structural characterizations [12 (link)–17 (link)]. SCiLS 2016b (Bruker Daltonics) imaging software was also used for further glycan expression analysis, comparison, and statistical evaluation (normalized to total ion current).

Imaging experiments were conducted using an ultrafleXtreme MALDI TOF/TOF with a 1 kHz Smartbeam laser (Bruker Daltonics, Billerica, MA, USA). Intact proteins were analyzed from 4,000 – 40,000 m/z with the global attenuator offset at 25%, and the lens adjusted to 7 kV. The digitizer was set to 0.08 Gs/s and the pulsed ion extraction was set to 50 ns. MSI was collected at 25 and 50 μm spatial resolution at 30 shots/spot, 1000 Hz, on medium energy distribution set to 75% laser power with hexagon measuring raster. Analysis of MALDI-MSI data was conducted in flexImaging, and flexAnalysis (Bruker Daltonics, Billerica, MA, USA) and SCiLS 2016b (Bremen, Germany). These are described in further detail on page S4 in the Supplementary Information.