Controlled Humidity Levels for Fine Spatial Detail Information in Enzyme-Assisted N-Glycan MALDI MSI

Investigation of the spatial distribution of N-glycans in tissue specimens has emerged as a powerful tool in clinical research, in part, because altered N-glycans are often a hallmark of disease progression. Mass spectrometry imaging of N-glycans relies on peptide N-glycanase spraying and tissue incubation for efficient in situ release of N-glycans from their carrier proteins. Unstandardized and uncontrolled incubation steps often cause significant delocalization of released N-glycans, resulting in the inability to link given N-glycan composition to a specific microanatomical region in the tissue. Herein, we optimized the incubation step to provide accurate and sensitive MALDI-MSI of N-glycans. Specifically, we tested saturated solutions of various salts that maintain constant relative humidity in the incubation chamber. We showed that the best performance was achieved using a saturated solution of KNO₃ that maintains an 89% RH. Under these conditions, near maximal sensitivity was achieved with the minutest ion delocalization, which we demonstrated at a 35 μm spatial resolution, where we observed six distinct spatial patterns that colocalize to distinct microanatomical compartments in a kidney nephrectomy tissue section.