N-glycan mass spectrometry imaging - a valuable tool for biomarker discovery and validation

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables spatially resolved detection of diverse biomolecules directly from tissues, supporting pathology-guided and unbiased biomarker discovery. By maintaining spatial context, MALDI-MSI overcomes key limitations of bulk omics approaches, which obscure the cellular origins of analytes, particularly in heterogeneous tumors containing abundant normal tissue or extracellular matrix. Complementary biofluids such as serum, urine, saliva, or cerebrospinal fluid are valuable for biomarker validation yet similarly lack spatial information when used as primary discovery materials. Integrating spatially defined diseased tissues with matched proximal biofluids therefore represents a powerful strategy for biomarker development. N-glycan mass spectrometry imaging (N-glycan MSI) has emerged as a versatile platform for characterizing N-glycan distributions in tissues and for quantifying glycan compositions in cells, antibodies, and biofluids. This method relies on on-tissue application of peptide N-glycosidase F (PNGase F) to liberate N-linked glycans, which are subsequently detected by MALDI-MSI to generate micron-scale, two-dimensional glycan maps. The workflow is highly modular, accommodating tissues, cultured cells, arrays, membranes, and isotopic or label-free quantification approaches. Recent advances also include the use of specialized glycosidases to resolve glycan isomers. This review highlights established and emerging N-glycan MSI strategies for biomarker discovery and validation, with applications in liver, brain, breast, and other clinically relevant tissues.