The Impact of the Mass Analyzer and Tissue Section Thickness on Spatial <i>N</i>-Glycomics with MALDI-MSI

We compared matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) spatial <i>N</i>-glycomics data sets from Fourier-transform ion cyclotron resonance (FTICR) and orthogonal accelerated time-of-flight (timsTOF) mass spectrometers of FFPE preserved human kidney samples. We also tested different tissue section thicknesses. In these analyses, we assessed the impact of the mass analyzer and tissue section thickness on <i>N</i>-glycan coverage, sensitivity, and histological alignment. Our results indicate negligible differences in <i>N</i>-glycan coverage between the two mass analyzers, where <i>N</i>-glycan annotation numbers remained consistent and were highly reproducible. The timsTOF-MS analyses demonstrated significant advantages with higher duty cycles and better lateral resolution, allowing for finer spatial resolution without compromising signal integrity. Specifically, timsTOF was able to generate detailed MALDI-MS images at 20 μm step size, accurately identifying <i>N</i>-glycan Hex:5 HexNAc:5 dHex:1 as a tubular-specific marker without observable delocalization. Despite minor annotation discrepancies, where only three species detected by FTICR were not detected by using timsTOF, and a few false-positive annotations from the timsTOF analysis attributed to lower mass resolving power, the overall consistency between the instruments was high. Importantly, tissue section thickness did not affect analysis sensitivity in the timsTOF analyses, with the average glycan signal intensity remaining stable between 7 and 2 μm sections. These findings demonstrate that 2 μm thick tissue slices can be effectively used in spatial <i>N</i>-glycomics workflows, maintaining sensitivity while enhancing confidence in pathohistological evaluations.