The Drake Lab

N-glycan MALDI imaging mass spectrometry

The original N-glycan imaging mass spectrometry method was developed in my laboratory, in collaboration with Dr. Anand Mehta. Since 2013, applications and utility of this method has continued to evolve and expand in collaboration with the Mehta and Angel laboratories at MUSC. As for other imaging mass spectrometry applications, the method bypasses the need for microdissection and solubilization of tissue proteins prior to analysis. The above schematic figure summarizes the method workflow. A key processing step is the spraying of a molecular coating of the enzyme that released N-glycans from their glycoprotein carriers, peptide N-glycosidase F (PNGaseF), using a solvent sprayer (TM Sprayer, HTX). As the reaction occurs in a humidified chamber, and not an immersed liquid solution, there is minimal diffusion of released N-glycans. Then, chemical matrix (CHCA; α-Cyano-4-hydroxycinnamic acid) is directly sprayed onto tissue sections, and soluble molecules are extracted from the tissue, and co-crystallized with the matrix. Protonated glycan ions are formed by irradiating the matrix with a pulsed laser and are separated by mass-to-charge ratio in a time-of-flight (TOF) mass analyzer, in the mass range of 700-4,000 Da. Spectra are collected across the tissue section in a grid pattern and used to generate two-dimensional molecular maps of up to 150 N-glycans directly from the surface of a tissue section. These molecular maps display the relative abundance and spatial distribution of the detected glycans, and when combined with histology and/or immunohistochemistry stains, glycan distribution can be correlated to known locations within a thin tissue section. 

MALDI MS imaging example of FFPE prostate cancer tissues can predict histology. A. H&E stain; B. Example tumor glycan; C. Example stromal glycan; D. Segmentation analysis distribution of 80 glycans. Images were processed in SCiLS Lab software.

Example N-linked glycan profiles from imaging mass spectrometry analysis of formalin fixed paraffin embedded prostate cancer tissues. Following PNGaseF release, N-glycans were detected using a high resolution 7T MALDI-FTICR mass spectrometer. Currently, including tissue microarray data, over 1000 PCa tissues ranging from benign hyperplasia to bone metastases have been analyzed. Unique glycan structural classes have been identified that are present in areas of inflammation and stroma, and on cancer cells as shown in two prostate tumor tissues (Figure 1, A-C). The bottom panels (Figure 1D) illustrate a segmentation analysis representation of glycan classes co-localized with histology features, representing the distribution of 80 N-glycans. This is a data analysis feature within SCiLS Lab software that is used routinely to analyze glycan distributions in tissues.

VIDEO: Using MALDI imaging to study protein glycosylation patterns in cancer