N-glycan imaging mass spectrometry (IMS) can rapidly and reproducibly identify changes in disease-associated N-linked glycosylation that are linked with histopathology features in standard formalin-fixed paraffin-embedded tissue samples. It can detect multiple N-glycans simultaneously and has been used to identify specific N-glycans and carbohydrate structural motifs as possible cancer biomarkers. Recent advancements in instrumentation and sample preparation are also discussed. The tissue N-glycan IMS workflow has been adapted to new glass slide–based assays for effective and rapid analysis of clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.
Imaging mass spectrometry (IMS) is a powerful tool to link changes in disease-associated N-linked glycosylation with histopathology features in standard formalin-fixed paraffin-embedded tissue samples.
N-glycan IMS has revealed specific carbohydrate structures and motifs associated with a variety of cancers with the potential for development into clinical biomarkers.
Advancements in instrumentation and sample preparation can effectively address N-glycan IMS limitations, such as sialic acid stability, structural isomer determination, and matrix effects.
The tissue N-glycan IMS workflow has been adapted to clinical biofluids, cultured cells, and immunoarray-captured glycoproteins for detection of changes in glycosylation associated with disease.
Protein glycosylation is a highly prevalent posttranslational modification involving the attachment of oligosaccharides, most commonly to a serine/threonine residue (O-linked glycosylation) or an asparagine residue (N-linked glycosylation). Glycosylation is a highly regulated process with more than 300 enzymes involved in their biosynthesis and processing in a non–template-driven manner. This process is done by a series of sequential reactions involving a wide array of glycotransferases that attach single monosaccharides from nucleotide sugar donors, as well as different glycosidases that remove individual monosaccharides. The expression, activity, and localization of these enzymes are sensitive to the physiologic state of the cell. Estimated to occur on more than half of human proteins, N-linked glycosylation has essential roles in protein folding, molecular trafficking, signal transduction, cell-cell interactions, and many other processes. Glycans serve as one of the initial points of contact during cell-to-cell interactions, so therefore disease changes in glycan biosynthesis can be more apparent than disease-related changes associated with gene mutations and proteins. Alterations in N-linked glycosylation have been found in a variety of diseases, including but not limited to cancer, inflammatory arthritis, liver fibrosis/cirrhosis, schizophrenia, type 2 diabetes, ischemic stroke, and Parkinson disease. Particularly for cancers, most US Food and Drug Administration–approved cancer biomarkers comprise circulating glycoproteins or carbohydrate antigens for measurement in blood. , , Glycoproteins can be ideal biomarkers because they enter circulation from tissues or blood cells through active secretion or leakage, making them assessable for analysis through serum. These current biomarker targets (eg, prostate-specific antigen or the carbohydrate antigen CA19-9) are far from ideal and have many limitations because of specificity and other factors.
There are many analytical approaches that have been applied to the analysis of disease-associated changes in the glycan constituents of glycoproteins in tissues biofluids. Reviews highlighting some of these methods are as follows, and include use of carbohydrate-binding lectins, , anti–carbohydrate antigen antibodies, glycan microarrays, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). Historically, there have been persistent challenges across all glycan analysis methods related to generally poor affinities of antibodies for carbohydrate epitopes, weak and nonspecific binding affinities of lectins, distinguishing isomeric species, and issues of extensive sample processing for HPLC and MS. The aforementioned reviews highlight progress in these areas, and the field in general is moving toward development of potential clinical and diagnostic assay workflows. Our group has recently developed several glycan targeted assays using imaging MS (IMS) approaches, including extensive detailed protocols. , Originally developed for spatial analysis of N-glycans in tissues, different adaptations of the approach have been developed for analysis of N-glycans of biofluids, cells, and captured proteins directly on glass slides. These approaches are distinguished by their overall throughput, ease and speed of preparation, and robustness. Using mass spectrometers equipped with matrix-assisted laser desorption/ionization (MALDI) sources for these assays, the consistent and reproducible detection of N-glycan structures in clinical samples has the potential to mature into clinical assays analogous to the MALDI MS–based assays that have transformed clinical microbiology identification of bacterial and other pathogen species. , The workflows, strengths, limitations, and clinical implications of using MALDI-IMS N-glycan analysis are discussed with examples for the analysis of tissues, clinical biofluids, cultured cells, and immunoarray-captured glycoproteins.