Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source

Abstract

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

Figure 1.

The new SubAP/MALDI-QEHF platform for N-glycan imaging. (a) Schematic illustration of the SubAP/MALDI-QEHF platform. (b) A snapshot showing the QEHF mass spectrometer integrated with the SubAP/MALDI source instead of an ESI source.

Figure 2.

Parameter optimization for N-glycan analysis on SubAP/MALDI MS platform (n=3). The TIC-normalized abundance (a), ion abundance (b) and TIC (c) of four representative N-glycans detected at different operating pressure. The TIC-normalized abundance (d), ion abundance (e) and TIC (f) of four representative N-glycans detected at different laser energy. The TIC-normalized abundance (g), ion abundance (h) and TIC (i) of four representative N-glycans detected at different laser energy.

Figure 3.

(a) More N-glycans were detected and imaged from FFPE mouse brain tissue section by using the novel SubAP/MALDI-MS platform. (b) The Venn diagram showing the overlap of N-glycans detected in this study with N-glycans reported in prior studies (Refs. 32 & 33) using vacuum MALDI-MS platform. (c) H&E stained FFPE mouse brain section post N-glycan imaging. (d-f) MS images of representative N-glycans detected from FFPE mouse tissue section. (g-h) Overlap of different N-glycan images clearly revealed different spatial distribution patterns of N-glycans on mouse brain section.

Figure 4.

N-glycans detected from FFPE mouse tissue section with ovarian cancer. The annotated glycan compositions were tentatively identified by searching against UniCarbKB database. H: Hexose; N; N-Acetyl glucosamine; F: Fucose.

Figure 5.

Images of N-glycans showing different spatial distribution patterns on FFPE mouse tissue section with ovarian cancer. (a) H&E stained FFPE mouse tissue section with ovarian cancer. (b-h) Complex N-glycans showed similar distribution in cancer area in comparison to peripheral area; (i-p) High mannose N-glycans accumulated in cancer area except Hex₃HexNAc₂.