MALDI Imaging of N-Glycans
Mass Spectrometry of Glycans
Glycans can be characterized and analyzed from basic monosaccharide units to intact glycan-lipid complexes using mass spectrometry. However, LC-MS/MS analyses typically require tissue homogenization. This can be an issue if a sample contains multiple tissue and cell types (ex. stromal cells, tumor cells, inflammation etc.). To combat this issue, lectins can be used for labelling of the glycan distribution within tissues. However, lack of specific sensitive commercial lectins has resulted in large limitations in the classification and spatial mapping of specific glycans within histological and histopathological samples. Because of this, MALDI imaging mass spectrometry (IMS) is the ideal method for examining the glycan profile and spatial distribution within a tissue.
The Method
Methods for using MALDI IMS to evaluate the N-glycome from FFPE tissue sections were first published in 2014 by Thomas Powers et. al. from the Drake laboratory. The developed workflow is amenable for evaluation of the vast number of stored FFPE clinical samples towards new studies in disease diagnosis and prognosis. This workflow reveals the complex carbohydrate interactions taking place within a specific FFPE tissue.
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“This workflow provides a valuable tool that allows for deeper investigations into the molecular causes of disease”
The Advantage
One advantage of using MALDI Imaging to analyze glycans is that their defined masses can be obtained and assessed reproducibly. Glycans in FFPE tissues make excellent targets due to their well-defined structures and low background. The signal is reproducible as it is generated from the activity of the PNGase F enzyme sprayed onto the tissue.
Example N-glycan signal from a single section of thyroid cancer. A) Total average spectrum from the image with example glycoform structures per m/z values. B) Tile view of example glycan images with unstained tissue prior to imaging.
“MALDI Imaging of N-glycans has allowed us to confirm that, for the most part, the same glycan and glycoprotein modifications we see in circulation are directly associated with the tumor and not adjacent tissue, or a distally adjacent tumor.””
Glycans DiffeR Throughout the Tumor Microenvironment
Quantification of N-glycan signatures from regional areas on a tissue. Statistical testing was done comparing nontumor adjacent and anaplastic regions (ROC-1) or anaplastic versus necrotic (ROC-2). A) Photomicrograph depicting areas selected and annotated by a pathologist for measuring the relative abundance of N-glycan expression. B) A high mannose glycan (Man9) structure distinguishing nontumor adjacent compared with anaplastic tumor or necrotic regions. C) A Biantennary N-glycan defining necrotic regions with low expression in nontumor adjacent and anaplastic regions. D) A tetrantennary fucosylated structure with low expression in adjacent nontumor and increasing expression in anaplastic and necrotic tissue regions.
In the above figure, tissue regions were selected from pathologist marked regions of adjacent nontumor, necrosis and anaplastic tumor to evaluate quantitative measurement of N-glycans. An example is shown of a high mannose structure (Man9) mapped to nonadjacent tissue areas with roughly a four fold increase based on median value compared to anaplastic tumor regions. Necrotic regions showed unique signatures apart from both nonadjacent tumor and anaplastic tumor regions. These examples illustrate that N-glycan expression is uniquely and quantifiably regulated within the tumor microenvironment, and these alterations can be detected by MALDI imaging of N-glycans.