Figure 1.
Schematic of the methodology for imaging N-glycans from FFPE tissues.
Prior to enzyme application, FFPE blocks are cut at 5 µm, incubated, deparaffinized and undergo antigen retrieval. PNGaseF is then applied and the slide is incubated before MALDI-IMS. The data is then linked with histopathology either on the same tissue slice or a serial tissue slice.
Figure 2.
MALDI-IMS of N-Glycans on Mouse Kidney Tissue.
Two mouse kidneys were sliced at 5 um prior to proceeding with the MALDI-IMS workflow. One tissue was covered with a glass slide during PNGaseF application to serve as an undigested control tissue. An average annotated spectra from the tissue that received PNGaseF application is provided (a). Tissue regions were assessed by H&E stain (b). The labeled peaks correspond to native N-glycans that have been reported for the mouse kidney on the Consortium for Functional Glycomics mouse kidney database. Two of these ions were selected and their tissue localization was assessed. Hex4dHex2HexNAc5 at m/z = 1996.7 (c) is located in the cortex and medulla while Hex5dHex2HexNAc5 m/z = 2158.7 (d) is more abundant in the cortex of the mouse kidney. An overlay image of these two masses is also shown (e), as well as the corresponding image from untreated PNGaseF control tissues (f).
Figure 3.
MALDI-IMS of a Human Pancreas FFPE Tissue Block.
An FFPE block of pancreatic tissue from a human patient was cut at 5 um prior to and selected for MALDI-IMS. Histopathology found four unique regions in the H&E of this tissue block. The tissue block contained tumor tissue, non-tumor tissue, fibroconnective tissue representing desmoplasia surrounding the tumor tissue, and necrotic tissue (b). MALDI-IMS was able to distinguish these four regions based off of specific ions after MALDI-IMS. M/z = 1891.80 (red) is found in the non-tumor (NT) region of the pancreas and corresponds to Hex3dHex1HexNAc6, while m/z = 1743.64 (blue) represents Hex8HexNAc2 and is predominant in the tumor region (T) of the tissue. Desmoplasia (DP) is represented by m/z = 1809.69 (green) corresponding to Hex5dHex1HexNAc4. In the region where necrosis was identified (TN), m/z = 1663.64 (orange) was elevated corresponding to Hex5HexNAc4. Image spectra were acquired at 200 µm raster. (c). Representative individual glycan images for the pancreatic FFPE tissue slice.
Figure 4.
MALDI-IMS of a Human Prostate FFPE Tissue Block.
An archived FFPE block of prostate tissue from a human patient was cut at 5 µm and prepared for MALDI-IMS glycan analysis, (a). H&E image. A global glycan imaging experiment performed with a raster of 225 µm demonstrated a heterogeneous expression of two glycan ions (b). at m/z = 1663.56 and (c). m/z = 1850.65. Stromal versus gland distribution were further assessed in a high resolution experiment at 50 µm raster (d–f). Column (d) indicates a 2× amplification of the H&E, and distribution of the same two glycans are shown at this magnification for m/z = 1663.56 (red) and m/z = 1850.65 (green), and an overlay image. Column (e) (enlargement of upper region shown in d). and (f) (enlargement of lower region shown in d), show two highlighted regions of stroma and glands enhanced at 10× resolution, with the same colors and glycans shown for column (d).
Figure 5.
Comparison of the Fragmentation Pattern of a Glycan Standard with the same Ion on Tissue.
(a). A representative MALDI spectra for native N-linked glycans from pancreatic cancer FFPE tissue. (b). NA2 glycan standard (m/z = 1663.6) was fragmented using CID, revealing a variety of cleavages across glycosidic bonds as demonstrated in the spectrum (a). When the same ion was fragmented on the pancreatic tissue, the fragmentation pattern was the same, verifying that we were detecting Hex5HexNAc4 in the human pancreas.
Figure 6.
N-Glycan Imaging of a Liver TMA.
A liver TMA purchased by BioChain consisting of 2 tumor tissue cores and one normal tissue core from 16 patients was imaged (200 µm raster). The H&E (a) provides the TMA location (red letters and numbers) and classifies whether the row is tumor (green bar) or non-tumor (red bar). M/z = 2393.95 (c) and m/z 1743.64 (d) were able to distinguish between hepatocellular carcinoma and uninvolved liver tissue. An overlay of these ion demonstrates that m/z = 2393.95 is elevated in tumor tissue and m/z = 1743.64 is elevated in normal tissue (b). Statistical data for these two ions is provided in Table 1.
Table 1.
Comparison of Tumor and Non-Tumor Glycans Detected in Hepatocellular Carcinoma Tissue Microarrays.