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Review
. 2021 Jul 29:15:711955.
doi: 10.3389/fnana.2021.711955. eCollection 2021.

Mass Spectrometry Imaging for Glycome in the Brain

Md Mahmudul Hasan  1 , Mst Afsana Mimi  1 , Md Al Mamun  1 , Ariful Islam  1 , A S M Waliullah  1 , Md Mahamodun Nabi  1 , Zinat Tamannaa  1 , Tomoaki Kahyo  1   2 , Mitsutoshi Setou  1   2   3
Affiliations
Review

Mass Spectrometry Imaging for Glycome in the Brain

Md Mahmudul Hasan et al. Front Neuroanat. .

Abstract

Glycans are diverse structured biomolecules that play crucial roles in various biological processes. Glycosylation, an enzymatic system through which various glycans are bound to proteins and lipids, is the most common and functionally crucial post-translational modification process. It is known to be associated with brain development, signal transduction, molecular trafficking, neurodegenerative disorders, psychopathologies, and brain cancers. Glycans in glycoproteins and glycolipids expressed in brain cells are involved in neuronal development, biological processes, and central nervous system maintenance. The composition and expression of glycans are known to change during those physiological processes. Therefore, imaging of glycans and the glycoconjugates in the brain regions has become a "hot" topic nowadays. Imaging techniques using lectins, antibodies, and chemical reporters are traditionally used for glycan detection. However, those techniques offer limited glycome detection. Mass spectrometry imaging (MSI) is an evolving field that combines mass spectrometry with histology allowing spatial and label-free visualization of molecules in the brain. In the last decades, several studies have employed MSI for glycome imaging in brain tissues. The current state of MSI uses on-tissue enzymatic digestion or chemical reaction to facilitate successful glycome imaging. Here, we reviewed the available literature that applied MSI techniques for glycome visualization and characterization in the brain. We also described the general methodologies for glycome MSI and discussed its potential use in the three-dimensional MSI in the brain.

Keywords: brain tissue; glycans; glycosylation; mass spectrometry imaging; three-dimensional MSI.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic workflow of mass spectrometry imaging (MSI) for glycome in the brain. An FFPE tissue section is mounted on an indium tin oxide-coated (ITO-coated) glass slide and dried by heating. Then section is deparaffinized and rehydrated by xylene and a series of ethanol rinsing, respectively. The epitope activation and tissue proteins are denatured by the treatment with a basic heat-induced antigen retrieval buffer (citraconic anhydride buffer, at pH 3) (Method-I). On the other hand, FF tissue (Method-II) rinses in an organic solvent for removing salts, lipids, and metabolites. Another FF tissue is directly mounted on the conductive ITO-coated glass slide or MALDI target plate for matrix spraying and MSI analysis (Method-III). Enzymatic release of N-glycans is performed using PNGase F under incubation at 37°C (Method-I). On-tissue derivatization is required (Method-I and II) before matrix deposition to stabilize glycans and enhance the ionization efficiency. After automated matrix spraying, the sections are subjected to a laser source for data acquisition and analysis.
FIGURE 2
FIGURE 2
Cleaving sites for enzymatic deglycosylation of glycans. N-glycan strategies (A) and O-glycan strategies (B). PNGase F cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless the core contains an α(1→3)-fucose. Cleaving site for PNGase F (A-i). N-glycosidase A hydrolyzes oligosaccharides containing a fucose residue α(1→3)-linked to the asparagine-linked N-acetylglucosamine (A-ii). Endo H cleaves the β-1,4 glycosidic linkage of high-mannose N-linked glycans between GlcNAc1 and GlcNAc2 (A-iii). In a sequential glycolytic cleavage, disialylated and trisialylated O-linked glycans have the sialic acid residues (NeuNAc) removed by α(2→3,6,8,9)-neuraminidase to release core-1 type O-glycans (B-i). Disialylated O-linked Core-2 hexasaccharide or sialic acid residues are sequentially degraded by α(2→3,6,8,9)-neuraminidase, β(1→4)-galactosidase and N-acetylglucosaminidase to release core-1 type O-glycans (B-ii). Cleavage site for O-glycosidase on the core-1 type O-glycans after sequential glycolytic degradation (B-iii).
FIGURE 3
FIGURE 3
Mass spectra and ion images for glycome in mouse brain tissues. (A) PNGase F digestion and MALDI-MSI analysis of N-glycans in mouse brain tissue. Extracted mass spectra of released N-glycans and matrix ions for (A-i) both, (A-ii) N-glycans and (A-iii) matrix. Representative ion images of (A-iv) Hex5HexNAc4Fuc1 at m/z 1809, (A-v) Hex7HexNAc2 at m/z 1581, (A-vi) Hex9HexNAc2 at m/z 1905, and (A-vii) shows the overlay image of m/z 1809 (red), m/z 1581 (green), and m/z 1905 (blue). Scale bar, 1.5 mm. The mass spectra (B-i) and ion images (B-ii) of gangliosides in the mouse brain coronal section without pre-treatment of the enzyme. Gangliosides are mostly localized in the cortex and hippocampus of the mouse brain tissue section using the CHCA matrix. Scale bar, 2 mm (upper panel), 500 μm (lower panel). The figures of panels (A,B) are reprinted and modified from Stanback et al. (2021) and Andres et al. (2020), respectively, with free-reuse permission.
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