Enhanced Detection of Charged N -Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging.

N -linked glycosylation represents a structurally diverse, complex, co- and posttranslational protein modification that bridges metabolism and cellular signaling. Consequently, aberrant protein glycosylation is a hallmark of most pathological scenarios. Due to their complex nature and non-template-driven synthesis, the analysis of glycans is faced with several challenges, underlining the need for new and improved analytical technologies. Spatial profiling of N -glycans through direct imaging on tissue sections reveals the regio-specific and/or disease pathology correlating tissue N -glycans that serve as a disease glycoprint for diagnosis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft hybrid ionization technique that has been used for diverse mass spectrometry imaging (MSI) applications. Here, we report the first spatial analysis of the brain N -linked glycans by IR-MALDESI MSI, leading to a significant increase in the detection of the brain N -sialoglycans. A formalin-fixed paraffin-embedded mouse brain tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of N -linked glycans. We report a comparative analysis of section thickness on the N -glycan detection using IR-MALDESI. One hundred thirty-six unique N -linked glycans were confidently identified in the brain tissue (with an additional 132 unique N -glycans, not reported in GlyConnect), where more than 50% contained sialic acid residues, which is approximately 3-fold higher than the previous reports. This work demonstrates the first application of IR-MALDESI in N -linked glycan imaging of the brain tissue, leading to a 2.5-fold increase in the in situ total brain N -glycan detection compared to the current gold standard of positive-mode matrix-assisted laser desorption/ionization mass spectrometry imaging. This is also the first report of the application of the MSI toward the identification of sulfoglycans in the rodent brain. Overall, IR-MALDESI-MSI presents a sensitive glycan detection platform to identify tissue-specific and/or disease-specific glycosignature in the brain while preserving the sialoglycans without any chemical derivatization.