Mass Spectrometry Imaging of N-Linked Glycans: Fundamentals and Recent Advances

Abstract

With implications in several medical conditions, N-linked glycosylation is one of the most important post-translation modifications present in all living organisms. Due to their non-template synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.

1. INTRODUCTION

This article is written in honor of Carlito Lebrilla, for all his outstanding and revolutionary contributions to the glycomics field (Charbonneau et al., 2016; Marcobal et al., 2011; Smilowitz et al., 2014; Zivkovic et al., 2011). Glycosylation is one of the most important post-translational modifications, as roughly 2% of the human genome encodes for glycosylation and half of all mammalian proteins are glycosylated (Apweiler, 1999). Glycans are carbohydrate-based polymers and can exist in several different forms. All glycan structures displayed in this review follow the Symbol Nomenclature for Glycans (SNFG) (Varki et al., 2015). A list of the most common sugar residues found on N-linked glycans and their corresponding SNFG is shown in Figure 1. The two main types of glycans that can be added to proteins are N-linked glycans and O-linked glycans (Figure 2). N-linked glycans are added to the asparagine residue in the N-X-S/T amino acid sequence, where “N” is asparagine, “X” is any amino acid except for proline, and “S/T” is either a serine or threonine residue. O-linked glycans bind to either an S or T residue. The majority of the glycan mass spectrometry imaging (MSI) literature investigates N-linked glycosylation. Therefore, N-linked glycan MSI is the focus of this review.

Figure 1.

Symbol Nomenclature for Glycans (SNFG) for common monosaccharides found in N-linked glycans.

Figure 2.

The two main types of glycans that bind to mammalian proteins. N-linked glycans bind to the N-X-S/T amino acid sequence, where “X” is any amino acid except for proline. O-linked glycans can bind to either a serine or threonine residue.

1.1. N-Linked Glycosylation

N-linked glycosylation plays several roles in physiological and pathological processes (Varki, 2006, 2008). N-linked glycans share a common chitobiose core which is composed of two N-acetylglucosamine (GlcNAc) residues and three mannose (Man) residues. This core, along with the N-X-S/T epitope, can be targeted by peptide-N-glycosidase F (PNGase F) to cleave N-linked glycans from glycoproteins. Unlike proteins, N-linked glycans follow a non-template synthesis (Figure 3). The process begins with the synthesis of the dolichol-linked precursor oligosaccharide in the endoplasmic reticulum (ER) (Kornfeld & Kornfeld, 1985). Then, an en bloc transfer of the oligosaccharide to a glycoprotein occurs in the ER before a series of enzymes trim off the glucose residues. The oligosaccharide then leaves the ER and enters the Golgi apparatus. In the Golgi, the outer mannose residues are removed from the Man₉GlcNAc₂ glycan before the GlcNAc-Galactose antennae are added. N-linked glycans can have two-four antennae. Sialic acid and fucose residues are added at the end before the glycoprotein leaves the Golgi. The spatial-temporal distribution of enzymes within the cell that act on the oligosaccharide to add sugar residues depends on the species, environmental factors, homeostasis, genetics and more (Varki et al., 2009). The final oligosaccharide can also have additional modifications such as a bisecting GlcNAc, which is where a GlcNAc residue is present on the center mannose residue of the chitobiose core between two antennae. Sulfate or phosphate modifications can also be implemented onto galactose (Gal) residues. Polylactosamine extensions, which consists of repeating GlcNAc-Gal units on the same antennae, are another type of modification. Once glycoprotein synthesis is complete, it will either leave the cell and join the extracellular matrix (ECM) or embed itself in the cell membrane.

Figure 3.

Non-template synthesis of N-linked glycans. The synthesis of the dolichol-linked precursor oligosaccharide occurs in the endoplasmic reticulum (ER). Then, the oligosaccharide is transferred to a protein and the glucose (Glc) residues are removed before exiting the ER and entering the Golgi apparatus. In the Golgi, glycans can undergo a series of modifications performed by a wide variety of enzymes. Once glycan synthesis is complete, the glycoprotein will either embed into the plasma membrane or join the extracellular matrix (ECM).

As a result of their non-template synthesis, N-linked glycan structures can be extraordinarily complex. Glycans vary in composition, structure, conformation, and linkage. However, they tend to fall into three main types of N-linked glycans: high mannose, complex, and hybrid glycans (Figure 4). High mannose glycans only contain Man residues outside of the chitobiose core and are prevalent in plants, fungi, and viruses. Complex glycans are composed of several modifications such as the addition of sialic acid and fucose residues. Hybrid glycans are a combination of high mannose and complex structures where one branch only contains Man residues, and the other contains different sugar residues such as Gal and sialic acid. A unique type of N-linked glycosylation is paucimannosylation. This type of glycan contains 2 GlcNAc residues, 1–3 Man residues, and a potential fucose residue. These complicated structural characteristics make analyzing glycans by mass spectrometry quite challenging, as oftentimes there are several possible structures for a given monoisotopic mass. MS₁ measurements can determine glycan composition, and MS/MS fragmentation can help elucidate structural and linkage information. Three-dimensional glycan features such as conformation and distinguishing between α- and β-linkages can only be determined using other analytical techniques such as ion mobility. While mass spectrometry alone cannot fully elucidate glycan structure, it is currently the most common analytical technique used to investigate glycans as it provides key insights into glycosylation patterns for a wide variety of biological samples.

Figure 4.

The three main types of N-linked glycans. High mannose glycans only contain mannose (Man) residues outside of the chitobiose core. Complex glycans can have different sugar modifications such as sialic acid and fucose residues. Hybrid glycans contain one branch with only Man residues and the other with complex residues.

1.2. Mass Spectrometry Imaging

First introduced by Richard Caprioli in 1997 (Caprioli et al., 1997), MSI is an analytical technique in which the spatial distribution of ions can be visualized within a biological tissue section. Each pixel or voxel in an ion heatmap corresponds to a mass spectrum that can contain hundreds or thousands of different ions. After entering a specific m/z ratio in the user’s data analysis software, the distribution of that ion’s m/z can be observed across the entire tissue section, thus creating the ion heatmap. The localization of analytes within a tissue is critical for many applications, especially in the clinical setting where certain analytes can be identified as disease biomarkers. Additionally, spatial information can help further potential drug therapies, especially for refining and developing those that target specific regions of the tissue.

There are several important factors to consider when designing a mass spectrometry imaging experiment (Xi et al., 2023). Sample preparation is especially important as the tissue’s morphological features must be preserved in order to retain spatial information. Fresh frozen (FF) tissue is a common type of tissue used in MSI as they require minimal sample preparation. The sample of interest is sectioned by a cryostat, mounted onto a glass slide and directly analyzed by MSI. If ionizing using matrix-assisted laser desorption ionization (MALDI), an energy-absorbing matrix is required to be evenly sprayed onto the tissue before MS analysis. Other chemicals, such as an internal standard for quantification, or enzymes that cleave specific glycan epitopes, may also need to be sprayed onto the tissue. Another important factor to consider for MSI experiments is spatial resolution (S. Ma et al., 2023; Wang et al., 2023). Step size and spot size are the two components that dictate an experiment’s spatial resolution (Figure 5). Step size is the distance between the centers of two different spots, and spot size is the diameter of the region that is ablated on the sample. Typically, the step size and spot size are the same measurement in order to minimize under- and oversampling. Smaller step sizes and spot sizes creates higher resolution images; however, higher resolution requires longer experiment times. Spatial resolution is primarily dictated by the wavelength of the laser used in the ionization source. For example, MALDI can achieve a spatial resolution of 1–2 microns using ultraviolet (UV) lasers, and IR-MALDESI can achieve 50 microns using infrared (IR) lasers (Joignant et al., 2022; Kompauer et al., 2017; Zavalin et al., 2012). Many techniques have been developed to improve spatial resolution, such as spraying certain matrices onto a tissue or utilizing complex optical trains. (Joignant et al., 2022; Kibbe et al., 2022).

Figure 5.

Spot size and step size determine spatial resolution in MSI. Spot size is the diameter of the ablation site after laser fire. Step size is the distance between the center of two spots. Smaller step sizes and spot sizes results in higher spatial resolution.

1.3. Mass Spectrometry Imaging of Glycans

N-linked glycan MSI allows for the spatial visualization of glycans within a biological tissue section and requires additional steps to consider beyond those required for traditional peptide and lipid imaging. While there are no official reporting standardizations in place, the glycomics community has created the Minimum Information Required for A Glycomics Experiment (MIRAGE), which contains essential guidelines to consider for multiple analytical platforms, including mass spectrometry (Kolarich et al., 2013; York et al., 2014). Although the analytical platforms in these guidelines do not include MSI, MIRAGE remains a valuable resource for many members of the community, especially those new to glycomics. With the recent rise in MSI technologies, it is expected that other types of glycans will soon be able to be imaged. This review will cover the fundamentals and recent advances of sample preparation, ionization mechanisms, instrumentation, data analysis, biological applications and methodological approaches for N-linked glycan MSI.

2. SAMPLE PREPARATION

Tissues are mostly composed of lipids and other metabolites. For example, 50% of the brain’s dry weight is composed of lipids (Hamilton et al., 2007). Since glycans are several orders of magnitude lower in abundance than lipids and other abundant metabolites, extensive sample preparation is required to ensure glycan signal is not suppressed by abundant metabolites (Drake et al., 2018). The best glycan signal has been observed in formalin-fixed paraffin embedded (FFPE) tissues. This is because the formalin-fixation process crosslinks amines, which are much more abundant in lipids and other metabolites compared to glycans (Puchtler & Meloan, 1985). Additionally, in the FFPE sample preparation protocol the dewaxing steps using xylenes and subsequent ethanol washes remove most of the lipids and metabolites in FFPE tissues. FFPE samples are typically used in histology laboratories and have many benefits compared to fresh frozen (FF) tissues such as improved preservation of morphological features, the ability to be stored at room temperature, and ease of sectioning. Importantly, FFPE tissues for glycan imaging are typically sectioned between 3–7 μm thickness for optimal enzymatic digestion and glycan (Drake et al., 2018).

2.1. Current Methods

The current sample preparation workflow used for N-linked glycan MSI experiments is the protocol developed by Richard Drake and Peggi Angel (Drake et al., 2018). FFPE tissue slides are heated in a 60°C oven for one hour to melt the paraffin wax surrounding and embedded into the tissue. Then, the tissue sections are treated to a series of xylenes, ethanol, and water washes to dewax and rehydrate the sample. Antigen retrieval is then used to undo the protein crosslinks formed during formalin-fixation. This step allows for more glycosylation sites to be exposed on the glycoproteins before pneumatic enzyme application using an automatic sprayer. PNGase F is typically the enzyme of choice for cleaving N-linked glycans from glycoproteins. Once the enzyme is applied to the tissue section, the sample slides will incubate for two hours at 37°C in a humidity chamber. The humidity chamber is prewarmed to >80% humidity which is required for maximized functionality of the enzyme (Rezaei et al., 2007). After enzymatic digestion, it is optimal to run the experiment within a few days after sample preparation to maintain sample freshness. The current protocol optimized for MALDI ionization has been adapted for multiple ionization platforms and has laid the foundation for many glycan imaging studies to come (Drake et al., 2018; Pace et al., 2022; Weigand et al., 2023). In addition to PNGase F, there are other enzymes that cleave glycans with specific features. These enzymes cleave between the GlcNAc residues within the chitobiose core, leaving behind a GlcNAc residue on the glycoprotein or glycopeptide. Endoglycosidase F3 (Endo F3) cleaves fucosylated biantennary glycans and tri-antennary glycans. Endoglycosidase F2 (Endo F2) cleaves complex biantennary glycans and some high mannose glycans. Endoglycosidase H (Endo H) cleaves high mannose and some hybrid glycans. Lastly, Endo D cleaves paucimannose glycans.

2.2. Improved Methods

There have been several studies aiming to improve the efficacy and efficiency of the glycan MSI sample preparation protocol. Each step in the protocol is crucial to ensure high glycan signal is detected, and omitting or inaccurately performing a step may result in complete loss of glycan signal (Drake et al., 2018). The humidity levels inside of the humidity chamber is one of the most important parameters for sample preparation, as adequate humidity levels are required for optimal enzymatic digestion. A study was conducted in which the incubation step was optimized in order to improve the accuracy and sensitivity of MALDI imaging of glycans (Veličković et al., 2022). Saturated solutions of different salts creating relative humidities ranging from 75–96% were tested to evaluate and identify the optimum relative humidity. It was found that a saturated solution of KNO₃ creating a relative humidity of 89% had the best balance between delocalization and sensitivity. This relative humidity also led to the highest number of N-linked glycan annotations (Veličković et al., 2022). Other studies have also reported the use of devices to determine the best balance between higher humidity and delocalization, with lower humidity and decreased enzyme efficiency by using micro-condensation (Fülöp et al., 2022). The implementation of these devices into laboratories would help standardize the enzyme digestion step within the glycan MSI community. In addition to humidity chambers, there has also been improvement in sample type coverage. As shown in Figure 6, Grgic and coworkers developed a sample preparation protocol for fresh frozen tissues that achieved glycan signal comparable to FFPE tissues (Grgic et al., 2023). Up until this point, FFPE tissues have been the gold standard for glycan MSI. However, this novel protocol improves the accessibility to glycan MSI, as glycan experiments are no longer limited to only those with access to FFPE tissues. Additionally, FF tissues do not require the extensive sample preparation involved with creating FFPE tissue blocks. There are a few key differences between the optimized FF protocol and the FFPE protocol. The FF protocol does not use xylenes washes, and the total number of washes was decreased. Additionally, ice-cold solvents were used. The antigen retrieval step, although typically only used for FFPE samples to undo protein crosslinks formed by formalin-fixation, was included as previous work showed that heating of the sample can lead to the denaturation of proteins which can expose more glycosylation sites (Høiem et al., 2022). Third, the incubation time was increased from two hours to eighteen hours (overnight), in addition to using the optimized KNO₃ saturated solution to maintain a stable relative humidity at 89%. While these methods have significantly improved sample preparation, there is room for expansion into other areas such as developing the protocol to analyze hard tissues, such as bones and plant, as this would vastly expand the glycan imaging field. Overall, much progress has been made in the effort to expand and improve the glycan MSI sample preparation, and it is likely the rapid advancement of automation will lead to further development.

Figure 6.

Comparable signal between fresh frozen tissue (top) and FFPE tissue (bottom) was achieved with the optimized FF protocol. N-linked glycan annotations are labeled with m/z value and signal-to-noise ratio (S/N). Permission obtained from Grgic et al., Sci Rep 2023, 13, 2776 © 2023 Springer Nature.

3. IONIZATION METHODS

The ionization source is one of the most important components of a mass spectrometer as all analytes of interest need to carry a charge for detection by the mass analyzer. The primary ionization mechanism used in MSI is matrix-assisted laser desorption electrospray ionization (MALDI). However, there are several other ionization mechanisms available for MSI, and compatibility with different types of mass spectrometers can vary. Glycans often have labile substituents, such as sialic acid, in which case a softer ionization source may be more favorable. However, chemical derivatization can be used to stabilize glycans to prevent fragmentation and loss of labile species during the ionization event. An overview of the ionization sources available for ionizing N-linked glycans for MSI is discussed herein.

3.1. MALDI

Matrix-assisted laser desorption ionization, or MALDI, is currently the most common ionization mechanism used in mass spectrometry imaging experiments, including for the detection of glycans (Figure 7). First developed by Karas and Hillenkamp in 1985, MALDI works by firing a laser at a sample section coated with an energy-absorbing matrix that will excite at the laser’s wavelength. (Karas et al., 1987, 1985). UV and IR wavelengths are the most common types of lasers used for MALDI. The use of a UV laser requires an energy absorbing matrix to be evenly sprayed across the sample before MS analysis. This can be done manually or by using an automatic sprayer. Additionally, MALDI can achieve a spatial resolution of 1–2 μm (Kompauer et al., 2017; Zavalin et al., 2012). While the theory behind the ionization mechanism is still up for debate, it is generally agreed upon tha18t the Lucky Survivor Theory and gas-phase protonation are taking place at the same time (Jaskolla & Karas, 2011). MALDI produces primarily singly-charged species and is not sensitive to negative ionization mode. Therefore, glycans typically receive their charge from a sodium adduct to create [M+Na]⁺ ions. This is because glycans prefer to adduct to the sodium ion in ambient sodium chloride instead of undergoing a protonation event (Gass et al., 2022). During the ionization event, MALDI deposits a higher amount of internal energy onto the sample compared to other ionization sources. Glycans that have labile substituents need to be chemically derivatized in order to prevent loss. Permethylation is the most common derivatization technique used for derivatizing N-linked glycans (Kameyama, 2021). It converts all the hydroxide groups on the monosaccharides to -OMe groups, rendering the glycans hydrophobic and significantly improving the ionization efficiency. In the event that sialic acid analysis is desired, an amidation or esterification reaction can be performed for chemical derivatization to stabilize the labile residue (de Haan et al., 2020; Liu et al., 2010; Nishikaze, 2019; Powell & Harvey, 1996; Reiding et al., 2014; Sekiya et al., 2005; Zhang et al., 2022).

Figure 7.

Matrix-Assisted Laser Desorption Ionization (MALDI). A UV-absorbing matrix is evenly applied to the sample before ionization. The laser beam fires at the sample causing the ablation of charged analytes and matrix molecules. Ions then head towards the mass spectrometer for detection.

3.2. MALDI-2

First developed in 2015, MALDI-2 is a laser-induced post ionization technique that demonstrates improved signal intensity for analytes ionized in negative mode polarity (Soltwisch et al., 2015) (Figure 8). After undergoing ionization by MALDI, the ions pass through a laser beam before entering the mass spectrometer (Dreisewerd et al., 2022). Although fully elucidating the ionization mechanism of MALDI-2 requires further research, the current theory consists of an interplay between resonance-enhanced two-photon ionization (REMPI), and conventional MALDI processes in large clusters and high post-ionization laser energies. Importantly, MALDI-2 can achieve a spatial resolution of 600 nm when operating in transmission mode (Niehaus et al., 2019). While it has been shown that MALDI-2 can improve ion yields in disaccharides (Soltwisch et al., 2015), a recent study reported the improvement in N-linked glycan signal in negative mode MALDI-2 compared to positive mode MALDI (Heijs et al., 2020). MALDI-2 improved the sensitivity of [M-H⁺]⁻ oligosaccharide species by three orders of magnitude compared to negative mode MALDI, and one order of magnitude compared to positive mode MALDI. Not only is negative mode ionization beneficial for the analysis of certain monosaccharides such as sialic acid, it can also eliminates the presence of multiple metal-adducted species for the same glycan. Due to the success of MALDI-2 in ionizing N-linked glycans, a protocol that discusses the guidelines for analyzing N-linked glycans using negative mode MALDI-2 was recently developed (Soltwisch & Heijs, 2023), allowing others to take advantage of the benefits MALDI-2 has to offer for N-linked glycan imaging.

Figure 8.

MALDI-2. The analytes undergo MALDI ionization before intercepting a laser beam and entering the mass analyzer. This laser-induced post ionization method showed significant improvement in negative ionization mode sensitivity for glycan mass spectrometry imaging, as well as for other metabolites.

3.3. DESI

Desorption electrospray ionization (DESI) is an ambient MSI ionization source that uses capillary tubes to fire an electrospray solvent containing charged droplets to desorb the sample spot before the resultant charged analytes head towards the inlet of the mass spectrometer (Claude et al., 2017) (Figure 9). Developed over 20 years ago, DESI imaging has been shown to be softer than MALDI ionization, and can analyze a diverse array of biological molecules including carbohydrates (Bereman et al., 2007; Morato & Cooks, 2023). Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization source that uses an additional capillary between the sample and mass spectrometer to facilitate the transfer of ions to the mass analyzer (Roach et al., 2010). Nano-DESI can achieve a spatial resolution down to 10 μm and can image a wide variety of molecules such as proteins, lipids, and metabolites (Yin et al., 2019). Unlike DESI, in a nano-DESI experiment the two capillaries form a liquid bridge on the sample surface. Recently, the first nanoDESI imaging of N-linked glycans was achieved (Weigand et al., 2023). A total of 38 N-linked glycans were detected in FFPE human prostate, and 34 in hepatocellular carcinoma (HCC). Sodiated species were detected in positive polarity, and deprotonated and chlorinated species were detected in negative polarity. Importantly, intact sialylated N-linked glycans were detected in the negative mode spectrum without chemical derivatization, indicating that nano-DESI is a soft ionization technique capable of preserving labile substituents on glycans.

Figure 9.

Desorption electrospray ionization (DESI). Electrospray solvent passes through a charged capillary to ablate the sample. Then, the desorbed analytes head towards the charged inlet of the mass spectrometer. DESI is an ambient ionization technique that can detect intact glycans without chemical derivatization and is sensitive in negative mode.

3.4. IR-MALDESI

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a hybrid and ambient ionization technique that combines the benefits of both MALDI and electrospray ionization (ESI) (Bagley et al., 2023) (Figure 10). IR-MALDESI begins by forming an ice matrix over the sample section. Then, an infrared laser with a 2.97 um wavelength is fired at the sample which will resonantly excite the water molecules present in the ice matrix and the sample. Currently, IR-MALDESI can achieve a spatial resolution down to 50 μm (Joignant et al., 2022). The ablated neutrals are met with an orthogonal electrospray plume and ionized via ESI-like mechanisms. Due to this ESI-like mechanism, IR-MALDESI is sensitive in negative ionization mode (Sampson et al., 2006). Additionally, IR-MALDESI is as soft as ESI, and N-linked glycans with labile sugar residues can be detected without chemical derivatization (Tu & Muddiman, 2019). As a result, IR-MALDESI has successfully detected intact sialylated N-linked glycans in several biological specimens (Pace et al., 2022; Pace & Muddiman, 2020). Glycans analyzed by IR-MALDESI are detected as multiply-charged species. Recently, a 5-fold increase in N-linked glycan detection was observed in rodent brain compared to prior MALDI reports (Samal et al., 2023). IR-MALDESI is also capable of predicting sialic acid content using the chloride adduction rule (Palomino & Muddiman, 2023). Using FFPE human kidney tissue, a distinct pattern was observed in which the number of sialic acids on a glycan was equal to the charge state minus the number of chlorine adducts. For example, with doubly-charged ions all glycans with two sialic acids were detected as [M-2H⁺]²⁻ ions, all glycans with one sialic acid were detected as [M-H⁺+Cl⁻]²⁻ ions, and all glycans without sialic acids were detected as [M+2Cl⁻]²⁻ ions. The source of chloride is the citraconic acid buffer which contains HCl to adjust the solution to ph~3.

Figure 10.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI). An ice matrix is formed over the sample prior to firing an IR-laser. The neutral analytes are then met by an orthogonal electrospray plume in which they pick up a charge before entering the mass analyzer. IR-MALDESI is also sensitive in negative ionization mode and detects sialylated glycans without the use of chemical derivatization.

4. INSTRUMENTATION

There are multiple parameters to consider when evaluating the type of mass spectrometer to use in a glycomics experiment. Resolution, resolving power, length of experiment time, and type of structural information the researcher wishes to achieve are just a few examples (Murray, 2022). There are mass spectrometers that come with a built-in ion mobility source, which can separate isomeric species (Dodds & Baker, 2019). Additionally, some mass spectrometers are limited in fragmentation (MS/MS) capabilities. Most instruments can perform MS², but not all can perform MSⁿ. Fragmentation strategies such as higher-energy collisional dissociation (HCD), collisional-induced dissocation (CID) and electron-transfer dissocation (ETD) are only available on certain types of mass spectrometers, but are beneficial in MSI studies for elucidating glycan structure (Hart-Smith, 2014; Kim & Pandey, 2012; Zubarev, 2004; Zubarev et al., 1998). Ultimately, the choice of mass spectrometer may come down to accessibility to instrumentation. Several types of mass spectrometers have been used for the analysis of N-linked glycans, and the two main types are discussed herein.

4.1. Time-of-Flight Mass Spectrometers

Time-of-Flight (TOF) mass spectrometers are one of the most common instruments used for analyzing N-linked glycans. There are several hybrid instruments such as quadrupole time-of-flight (QToF) and TOF-TOF which introduce tandem MS capabilities. Additionally, some commercialized TOF instruments come with an ion mobility source which would separate isobaric and isomeric species (Li et al., 2023). TOF instruments work by giving all ions the same kinetic energy, and the ions will “fly” the distance of the flight tube. The detector measures the arrival time of all ions, and this is a function of the m/z ratio as larger ions will take longer time to get to the detector. Additionally, ions with higher charge states will fly faster as the electrical field has a stronger effect on them. As a result, TOF has a theoretically unlimited mass range, which is beneficial for glycomics as glycans can become very large structures (J. Lee & Reilly, 2011). Larger N-linked glycan structures consist of multiply-branched structures and structures with polylactosamine extensions. Another benefit of TOF instruments is the high spectral acquisition rate which can be up to 10,000 spectra per second. The main downside to TOF instruments is low resolving power, as it routinely achieves 30,000 RP at full width half max (FWHM), which could complicate data analysis if there are multiple glycans with similar masses. Importantly, when performing MALDI-TOF charged slides are required to help facilitate ion production of which indium-tin-oxide (ITO) coated slides are the most common (Mezger et al., 2021). Overall, the TOF is a powerful instrument that has been used for decades and continues to be a valuable resource for the analysis of N-linked glycans.

4.2. High Resolution Accurate Mass Instrumentation

High resolution accurate mass (HRAM) instruments can achieve high resolving power (RP) and mass measurement accuracy (MMA). RP is defined as the ability of the mass spectrometer to define two neighboring peaks (Murray, 2022). MMA is the difference between the theoretical and experimental m/z and is typically reported in parts-per-million (ppm). The two most common types of HRAM instruments are the Orbitrap and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass analyzers (Makarov et al., 2009; Marshall, 1979). Most modern Orbitraps have a quadrupole that act as a mass filter to only send ions within the user specified mass range to the detector. Other Orbitraps can come with an ion trap, and there are also tribrid instruments that contain the Orbitrap, quadrupole, and ion trap. The main benefits of the Orbitrap include high resolving power (480,000 at 200 m/z regularly achieved), high MMA, space charge capacity, and transmission. Orbitrap technology uses a central spindle to separate different m/z ratios. Ions are electrodynamically squeezed into different ion packets that vary by their m/z ratio. Both Orbitrap and FT-ICR use image current detection that is fast Fourier transformed to wavelength, which is then converted to m/z. In the FT-ICR, ions are injected into the ICR cell containing a high magnetic field. Lower mass ions will circulate at a higher frequency than higher mass ions, and this frequency is measured by the detector plates. Currently, the FT-ICR achieves the highest MMA and resolving power (1,000,000) of any mass analyzer. The main disadvantage of FT-ICR is the maintenance and safety concerns involved with super conducting magnets. Overall, HRAM instruments can detect N-linked glycans with high MMA, which is especially important in MSI as most studies rely on accurate mass measurements for glycan annotations.

5. DATA ANALYSIS

There are vendor-specific and vendor-neutral software available for processing MSI data sets. These software can perform a wide variety of functions, including statistical analyses and overlaying stained tissue sections onto an MSI file to visualize the colocalization of analytes with the tissue’s morphological features. MSI data files are typically stored in the imzML format as most MSI software tools support this file type (Schramm et al., 2012). However, there is currently no software that can automatically annotate glycans. Therefore, most annotations are done manually using raw files collected by the mass spectrometer and a glycan database. There are three major glycan database projects that all emerged roughly around the same time, and to make sure that each project had its own unique purpose, the GlySpace Alliance was formed. One of the members of this coalition is Glyco@Expasy which was first released in 2016 (Mariethoz et al., 2018). This portal houses GlycoMod and GlyConnect: two useful bioinformatic tools in N-linked glycan annotation (Alocci et al., 2019; Cooper et al., 2001). GlycoMod produces glycan compositions based on a user-inputted mass (Cooper et al., 2001). Some compositions may have a direct link to the GlyConnect database, which provides experimentally determined glycan structures previously reported in the literature (Alocci et al., 2019). Another member of GlySpace Alliance is GlyCosmos which was founded in Japan (Yamada et al., 2020). GlyCosmos contains data and information relating to all fields of the glycosciences, including glycan-related diseases. It also houses GlyToucan, an international glycan structure repository (Tiemeyer et al., 2017). The last member of GlySpace Alliance is GlyGen, which first began in 2019 (York et al., 2019). GlyGen combines information from other biological disciplines and methods, allowing for a comprehensive analysis of glycosylation and related biology in the tissue (York et al., 2019). Tandem mass spectrometry produces a different type of dataset which requires different bioinformatic tools. GlycoWorkbench (GWB) is software that can annotate glycan structures, but also has specials tools to annotate glycan fragments from MS/MS spectra (Ceroni et al., 2008). This can help confirm glycan structure, as GWB can calculate theoretical glycosidic and cross-ring fragments for a glycan drawn in the canvas. All of these glycan bioinformatic resources are freely available online, and a summary of all software and their benefits is listed in Table 1.

Table 1.

A summary of bioinformatic tools available for glycan MSI data analysis. All portals and software are freely available online.

Software	Key Benefits	Reference
GlyConnect	An experimentally curated database with glycan structures. Many different types of species are represented in the database.	(Alocci et al., 2019)
GlyCosmos	A portal containing data and information regarding the glycosciences and all glycan-related biology such as genes, lectins, and diseases.	(Yamada et al., 2020)
GlycoMod	A database that outputs theoretical glycan compositions for an inputted mass. It can also be used for glycoproteomic analysis.	(Cooper et al., 2001)
GlyGen	Combines glycoscience data and information with other related biological disciplines and -omics.	(York et al., 2019)
GlyToucan	Repository for glycan structures available in GlyCosmos.	(Tiemeyer et al., 2017)
GlycoWorkbench	Produces theoretical fragments of glycans (both glycosidic and cross-ring fragments) to help annotate glycan fragmentation spectra.	(Ceroni et al., 2008)

There has also been recent emergence of novel data analysis tools in the past few years, particular in the glycan database realm. METASPACE is one of the most popular MSI automatic annotation platforms that can generate analyte annotations at a fixed false discovery rate (FDR). METASPACE generates annotations using spatial chaos, spectral isotope, and spatial isotope parameters. Recently, an N-linked glycan database, NGlycDB, was integrated into METASPACE (Veličković et al., 2021). Using N-linked glycans reported on GlyConnect, the database was created to be compatible with METASPACE. Users can upload imzML files directly into METASPACE, specify the FDR, and a list of potential glycan molecular formulas will be generated. Once a molecular formula is selected, users can visualize the spatial distribution and see all potential compositions and SNFG structures. In addition to the novel METASPACE database, an N-glycome atlas was recently developed which analyzed distinct differences in N-linked glycosylation between healthy and cancerous tissues for 15 different tissue types using MALDI-MSI (Wallace et al., 2024). Figure 11 shows the 15 tissues that were analyzed and the corresponding glycan that was most altered in cancer. A tissue microarray (TMA) was used in addition to FFPE tissue glycan imaging for this comprehensive study. Changes in N-linked glycans, core fucosylation, and sialylation between healthy and cancerous tissues for 15 different tissues was described, providing the foundation for future investigations into these devastating cancers. All data from this study was deposited into METASPACE, and detected glycans were linked to the GlyToucan repository in GlyCosmos. There has been significant improvement in the data analysis of N-linked glycan MSI, with the development of novel databases and advancements of technology, automated annotation of N-linked glycans is soon to come.

Figure 11.

Spatial distributions of N-linked glycans that were most altered in cancer. Putative glycan structures highlighted in red demonstrated increased abundance in cancer, and structures highlighted in green demonstrated decreased abundance in cancer. (A) Bladder, (B) breast, (C) cervix, (D) colon, (E) esophagus, (F) gastric, (G) kidney, (H) liver, (I) lung, (J) pancreas, (K) prostate, (L) sarcoma, (M) skin, (N) thyroid, (O) uterus. Permission obtained from Wallace et al., Sci Rep 2024, 14, 489 © 2024 Springer Nature.

6. BIOLOGICAL APPLICATIONS

MSI is one of the most powerful tools to investigate disease (Arentz et al., 2017) because the spatial distribution of metabolites within a tissue allows researchers to determine disease-related changes within specific regions of the tissue. Changes in N-linked glycosylation have been implicated in several diseases. The most common disease that has been investigated using N-linked glycan imaging is cancer, as N-linked glycans have been shown to play roles in tumor prognosis and are an established biomarker in cancer progression (Taniguchi & Kizuka, 2015). However, there are several other diseases that are implicated with changes in N-linked glycosylation. This section will highlight recent studies on cancer, neurological disorders, liver disease, and other important biological diseases.

6.1. Cancer

Changes in N-linked glycosylation have been implicated in many forms of cancer. An increase in high mannose N-linked glycans and core fucosylation has also been observed in several cancers (McDowell et al., 2023). Other glycan features such as branching and the presence of a bisecting GlcNAc have also been shown to be elevated in cancerous tissues (Wallace et al., 2024). Most notably, an increase in sialylation is implicated in almost all types of cancer. As such, it is important to understand the spatial distribution of the sialylated glycans within the tissue (Ma & Fernández, 2022). A comprehensive overview of changes implicated in cancer N-linked glycosylation has been recently established (McDowell et al., 2023). Recently, a study on endometrial cancer reported an increase in high mannose N-linked glycan structures and a decrease in complex structures in the tumor regions of cancerous endometrial tissue (Mittal et al., 2021). Structures were elucidated using a tissue microarray and porous graphitized carbon liquid chromatography-MS/MS (PGC-LC-MS/MS). This was the first study to investigate FFPE endometrial cancer (EC) tissue using MALDI-MSI. Additionally, N-linked glycosylation between EC tumor regions with lymph node metastasis (LNM) and without LNM were compared, and a core fucosylated glycan (Hex₂HexNAc₂Deoxyhexose₁+Man₃GlcNAc₂) was found at higher abundances in the primary tumor region of EC tissues associated with LNM. Another recent study investigated N-linked glycosylation in colorectal cancer, in which a decrease in high mannose and bisecting glycans, and an increase in paucimannose biantennary, hybrid, and tetra-antennary glycans was observed in cancerous tissues using MALDI (Figure 12) (Young et al., 2024). Each stage of this disease was investigated, with distinct differences in glycosylation reported at each stage of progression. Another study also investigated colorectal cancer at each stage of the disease, in which it was found that high mannose glycans were at the highest abundance in pre-malignant tissues (Boyaval et al., 2022). MALDI MSI was used in conjunction with capillary electrophoresis (CE) ESI-MS/MS in order to elucidate glycan structure.

Figure 12.

(A) Spatial distributions of glycans in colorectal tissue represented by segmentation analysis. Increased paucimannose glycans (B), tetra-antennary glycans (C), and bi-antennary glycans (D) was observed in cancerous tissue. Decreased high mannose glycans (E) and bisecting glycans (F) was observed in the cancerous tissue compared to normal tissue. Tumor associated polyp (TAP). Tubulovillous adenoma (TVA). Permission obtained from Young et al., Front. Pharmacol. 2024, 14 © 2024 Young, Nietert, Stubler, Kittrell, Grimsley, Lewin, Mehta, Hajar, Wang, O’Quinn, Angel, Wallace, and Drake.

There was a study investigated prostate cancer in over 100 patients using TMAs, which allowed for high-throughput analysis of FFPE tissues (Conroy, Stanback, et al., 2021). It was found that diseased patients had an increase in high mannose, and tri- and tetra-antennary glycans with core fucose residues. Interestingly, bisecting, sialylated and core-fucosylated glycans were elevated in benign tissues compared to prostate tumors, in contrast to the trend observed in many other cancerous tissues. This study also investigated differences in prostate tumors between white and black patients, and it was found that branched, bisecting, and biantennary glycans were higher in the black patient cohort. Studies that look at differences between racial groups are essential to addressing ongoing health disparities, since minorities have poorer patient outcomes compared to the white population (Kawachi et al., 2005). Another study investigated epithelial ovarian cancer (EOC) specifically looking at sialylated glycans within the tissue as prior literature focused on neutral, non-sialylated glycans (Grzeski et al., 2022). Tumors had elevated levels of α2,6 linked sialic acid, while adjacent tumor stroma had an abundance of α2,3 linked sialylated glycans. Since this study used MALDI ionization, chemical derivatization of sialic acid was used. N-linked glycosylation in other gynecological cancers has also been investigated using MSI. For example, breast cancer tissues have elevated polylactosamines and sialyation (Scott et al., 2019; Scott & Drake, 2019). Recently, it was discovered that high mannose and fucosylated glycans increase in abundance with breast cancer progression (Ščupáková et al., 2021). Lastly, another study was conducted using 2.5D MSI to investigate esophageal cancer (Vos et al., 2022). First, 3D MSI was conducted on 24 serial sections of FFPE tissue to determine the minimum number of sections required to visualize tissue heterogeneity at each stage of the disease. From this, a subset of 4 sections was used to analyze esophageal cancer at each stage. These 4 sections are what is called 2.5D MSI, in which an increase in specific high mannose and complex glycans was detected in the later stages of esophageal cancer. Overall, MSI can measure distinct changes in N-linked glycosylation in malignant tissues and has had a significant impact on cancer research as it continues to drive forward our understanding of cancer glycobiology.

6.2. Lung Disease

The COVID-19 pandemic first emerged in December 2019 and has caused the loss of millions of lives since (Ciotti et al., 2020). Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the viral pathogen that causes coronavirus disease (COVID-19). The severity of the disease in patients heavily depends on the efficacy of the patient’s immune response, which warrants investigation of glycosylation patterns in COVID-19 infected tissues. A recent study used MALDI MSI and immunohistochemistry (IHC) to image human lung autopsy tissues to correlate the spatial distribution of N-linked glycans to both virus and immune cells that have infiltrated the tissue (Jones et al., 2022). Three different tissues representing different amounts of time spent on a ventilator (0, 2, and 27 days) were highlighted in this work. In all tissues, core-fucosylated and tetra-antennary glycans localized to CD163+ immune cells. Interestingly, the colocalization of high mannose glycans with CD8+ and CD11+ immune cells was observed in the tissues that spent 0 and 27 days on a ventilator. This likely indicates a metabolic switch from oxidative phosphorylation to glycolysis in order to regulate CD8+ cells (Buck et al., 2015; Demotte et al., 2008; Sukumar et al., 2013). Chemical derivatization of sialic acid was performed in order to differentiate sialic acid linkages within the tissue to elucidate anomeric configuration. Across the tissue that spent 2 days on a ventilator, a biantennary α2,6 sialylated glycan was most abundant (Figure 13A,C). Additionally, the sialylated glycans colocalized to the CD8+ and CD11+ immune cell regions were primarily α2,6 linked (Figure 13F,G). In conclusion, MSI and IHC were able to colocalize different types of N-linked glycans to specific cell clusters on human lung tissue, and ongoing research includes connecting these findings to the metabolic activity within the tissue.

Figure 13.

(A-C) Spatial distributions of sialylated biantennary glycans and (D) an overlay image of all three glycans demonstrating spatial overlap. (E) Highlighted region demonstrating an overlay of glycans in (B) and (C). (F) Composite image of sialylated glycans in (E) overlaid with α2,6 disialylated bisecting glycan specific to the CD8+ regions. (G) Composite image of (E) overlaid with α2,6 sialylated tetra-antennary complex glycan localizing to CD11b+ region. Permission obtained from Jones et al., Front. Anal. Sci. 2022, 2:1021008 © 2022 Jones, Drake, Dressman, Parihar, Stubler, Masters and Mercer.

6.3. Neurological Disorders

The glycan signature of the brain is substantially different from other tissues in the body, and therefore, the investigation of N-linked glycosylation within neurological systems has been on the rise in recent years (Conroy, Hawkinson, et al., 2021; Williams et al., 2022). The advancement of MSI techniques enables researchers to investigate differences in glycosylation between different regions of the brain such as the corpus collosum, striatum, and cerebral cortex, all of which have unique and important roles in brain function. There have been recent reports investigating the healthy brain, in which high sialic acid content was detected in all regions of the brain (Samal et al., 2023). Importantly, there are also distinct differences between healthy and diseased brain tissue. Neuroinflammation occurs in injuries and neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), and recently it was shown that neuroinflammation results in an increase in high mannose and bisecting glycans, and a decrease in sialylated and core fucosylated glycans (Rebelo et al., 2021). These experiments were conducted using a lipopolysaccharide (LPS) injection into rodent brain and MSI was performed using MALDI. In Alzheimer’s disease, increased N-linked glycosylation was observed in both the frontal cortex and hippocampus of rodent brain (Hawkinson et al., 2022). Increases in N-linked glycosylation was only observed in the frontal cortex in human brain. A significant decrease of N-linked glycans was observed in the hippocampus region, which is a stark contrast to AD in rodent brain (Figure 14). In another recent study, N-linked glycosylation changed in a region specific manner within PD brain (Rebelo et al., 2023). Neutral glycans were seen to have significantly decreased in PD brain compared to healthy brain, while both core and outer arm fucosylation increased in PD brain. However, polysialic acids (PSA) were elevated in the striatum region of the brain, but decreased in the substantia nigra (Rebelo et al., 2023). Additionally, branching also varied between each brain region. N-linked glycans with two branches increased in the striatum but decreased in the substantia nigra. Tri-antennary glycans were upregulated in both regions, while tetra-antennary glycans significantly decreased in the striatum but increased in the substantia nigra. In summary, the brain is an incredibly complex system with several different regions each performing important functions for the body. This necessitates detailed investigation into the glycosignature of all brain regions in order to understand the hidden glycobiology behind the progression of neurological disorders.

Figure 14.

(A) Total ion chromatogram of human frontal cortex (top) and whole mouse brain (bottom) showing detected N-linked glycans. (B) Similarities and differences in changes in N-linked glycosylation of AD between human and mouse frontal cortex and hippocampus. Permission obtained from Hawkinson et al., Alzheimer’s & Dementia. 2021, 18(10), 1721–1735 © 2021 John Wiley & Sons, Inc.

6.4. Liver Disease

The liver is one of the most important organs, responsible for filtering all of the blood in the body and producing bile (Kalra et al., 2024). Unfortunately, there are diseases that can inhibit these important processes, such as Hepatitis A, B, and C. Another common liver disease is metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease. A recent study used a new derivatization strategy to stabilize sialic acid within the livers of healthy mice and mice with the progressive form of liver disease (Saito et al., 2021). Sialic acids were derivatized with benzylamine, which produced higher intensity signals compared to previous derivatization strategies. Additionally, increased sialic acid content and fucosylated glycans were detected in the fatty liver disease mouse liver model compared to healthy mouse liver. A separate study investigated the effect of this liver disease using a mouse model and human liver samples (Ochoa-Rios et al., 2022). The mice used in the mouse model were fed a high fat diet (Western diet), and after 18 months the mice were sacrificed, and the livers were analyzed using MALDI-MSI. Multiple types of glycans, such as high mannose, complex, fucosylated, and hybrid glycans were elevated in the diseased liver mouse tissue compared to healthy mice. Human liver samples with MASLD were also obtained and evaluated by MALDI-MSI, and similar results were observed using MALDI-MSI. The increased levels of glycans localized to the new fatty areas of the liver tissue that were not present in healthy liver tissue. Additionally, core-fucosylated glycans were shown to be caused by the fibrosis present in the liver diseased tissue. A follow-up study investigation was conducted in which N-glycosylation was quantified within each stage of MASLD progression (Ochoa-Rios et al., 2024). Interestingly, early stages of MASLD had an increase in fucosylation and branching, which has also been shown to be elevated in liver cancer. Across all of these studies, an increase in fucosylation was observed in diseased tissues, suggesting that potential drug therapies can be developed to target fucosylated regions of the liver. Overall, N-linked glycan MSI reveals the importance of uncovering changes in glycosylation within diseased tissues, especially in major organ systems such as the liver.

6.5. Other Diseases

There are several other diseases that have been investigated using N-linked glycan MSI. Osteoarthritis, specifically knee osteoarthritis (KOA), was analyzed using MALDI-MSI (Y.-R. Lee et al., 2022, 2023a, 2023b), finding elevated levels of complex N-linked glycans in KOA FFPE tissue compared to healthy tissue. Differences in glycosylation were detected between lateral and medial cartilage, and MS/MS was performed to structurally elucidate the glycans. Three complex glycans and one high mannose glycan were found to be elevated in the medial cartilage compared to lateral cartilage, and fucosylated glycans were elevated in the lateral cartilage compared to medial cartilage. Another study investigated congenital aortic valve stenosis (AS), where the aortic valve narrows and prevents blood flow. They examined the spatial distribution of N-linked glycans within the aortic valve tissue (Angel et al., 2021) and saw sialylated glycans were elevated in the AS tissues compared to normal aortic valve. Core fucosylated glycans showed distinct localization within normal tissues, but completely different localization patterns in AS tissues. This was the first study to investigate N-linked glycosylation within the aortic heart valve and demonstrated the significant impact that AS has on the distribution and regulation of N-linked glycans in the tissue. An investigation of canine gliomas, malignant tumors that form on the brain and spinal cord, with N-linked glycan MSI (Malaker et al., 2022) revealed that sialylated glycans were localized to the necrotic regions of the gliomas. These glycans were detected with sodium and potassium adducts, and adjacent glycoproteomic analysis complemented these annotations. Other types of sialylated glycans (O- and S-linked glycans) were also detected using glycoproteomics. A human larynx was imaged recently using high resolution MALDI-MSI and magnetic resonance imaging (MRI) (Kishimoto et al., 2021). This patient had a mucus retention cyst form on the larynx, and several complex N-linked glycans localized to the cyst region. This was the first study to perform N-linked glycan MSI on human larynx. Finally, a study was conducted to investigate the spatial distribution of N-linked glycans within beef, mutton, and pork tenderloin (Guo et al., 2023). Allergic reactions to meat occur after tick bites due to the galactose-galactose unit present on glycoproteins in meat. In this analysis, it was found that N-linked glycans with the galactose-galactose residue present on the terminal end were most abundant in the fibroconnective tissue of the meat, and not present in the actual meat fibers. This finding shows that processed meat, such as sausages or canned meats, can be safe to consume by individuals who have been bitten by a tick as these meats consist of only meat fibers. Although N-linked glycan MSI has already shown significant coverage of several different pathologies, with the ease and availability of FFPE tissues and now fresh frozen tissues, it will continue to expand to other diseases and reveal distinct glycosylation patterns yet to be discovered.

7. NOVEL AND IMPROVED METHODOLOGICAL APPROACHES

While the sample preparation protocol has become quite standardized for N-linked glycan MSI, there have been several contributions to the literature that cover novel approaches for improving certain aspects regarding glycan MSI. Most studies aim to improve molecular coverage and acquisition time, while other studies attempt to circumvent the limitations brought about by MSI, such as the inability to distinguish isomers. This section will discuss the recent advancements in regard to these approaches.

7.1. Multiplex, Multimodal, and Multiomic MSI

Multiplexing is when multiple types of ions can be visualized and analyzed within the same experiment, and it is most often conducted using LC-MS (Chapman et al., 2014). In MSI, multiplexing allows for different types of biological molecules to be spatially visualized concurrently. As a result, this methodology shortens experimental acquisition time and maximizes the amount of information that can be extracted from one biological tissue section. It was recently reported that N-linked glycans and glycogen can be multiplexed into one assay by using MALDI-MSI and ion mobility (Young et al., 2022). Glycogen is made of glucose polymers and serves as the primary way to store glucose in living organisms. This study treated FFPE tissue sections with isoamylase and PNGase F to release glucose polymers and N-linked glycans, respectively. With the help of traveling wave ion mobility separation (TW-IMS), the colocalization of glucose polymers and N-linked glycans was able to be observed and analyzed within the FFPE tissue section. An additional study multiplexed collagen peptides and N-linked glycans using MALDI-MSI and coupled this to single cell spatial omics (Dunne et al., 2023). Different antibody-based single cell workflows were evaluated for their performance before and after MALDI-MSI multiplexed analysis. Overall, it was determined that these workflows can be successfully performed alongside MALDI-MSI analysis on the same tissue section, allowing for maximized spatial information of both cellular and extracellular modalities. This study is also an example of multimodal MSI, which is the use of multiple methods to spatially visualize molecules. The use of MALDI-IHC, which can be employed alongside MSI to achieve higher plex capabilities, can also be used to colocalize labeled biomolecules to unlabeled species. While these studies are all examples of multiomic MSI, there was another study conducted that used the same tissue section to image lipids, N-linked glycans, and tryptic peptides (Figure 15) (Denti et al., 2022). First, a MALDI matrix was sprayed before lipid MSI analysis. After the matrix spray removal, PNGase F was sprayed onto the slide, incubated for enzymatic digestion, and N-linked glycan MSI is performed. This cycle is repeated with trypsin enzyme in order to perform tryptic peptide MSI. The study found that all three of these analytes were able to be imaged and visualized on the same tissue section, increasing the amount of spatial information that can be obtained from potentially crucial and limited samples. The incorporation of additional techniques and methodology has allowed for scientists to cover multiple facets of biology in addition to glycosylation, and the rapid advancement of technologies will continue to allow researchers to uncover more information from complex and limited biological samples.

Figure 15.

Schematic of sample preparation methodology for sequential imaging of (A) lipids, (B) N-linked glycans and (C) peptides using a single FFPE tissue section. Permission obtained from Denti et al., J. Proteome Res. 2023, 22(6), 2149 © 2021 Denti, Capitoli, Piga, Clerici, Pagani, Criscuolo, Bindi, Principi, Chinello, Paglia, Magni and Smith.

7.2. Enhanced Detection and Isomer Differentiation

One of the major limitations of glycan MSI is that glycans need to compete for ionization against abundant metabolites present in the tissue. Unlike LC-MS, separation apparatuses that help enhance glycan signal such as molecular weight cut off (MWCO) filters or porous graphitized carbon solid phase extraction (PGC-SPE) which separate polar analytes from nonpolar analytes cannot be used because the tissue section cannot be homogenized for MSI. Therefore, it is imperative that glycans are sufficiently cleaved from proteins and maximum MS signal is achieved. A recent study showed that N-linked glycan signal improved 6-fold using gelatin-coated ITO slides compared to conventional ITO slides when analyzing bone-cartilage tissue (Y.-R. Lee et al., 2021). Due to the decalcification process, cartilage-bone tissue has difficulties adhering to conventional ITO slides. However, by lowering the temperature of antigen retrieval and increasing incubation time, not only did glycan signal increase but so did the number of glycans detected in cartilage-bone tissue mounted on gelatin-coated ITO slides. Another method that was recently reported enhanced the detection of non-sialylated glycans using MALDI MSI and a combined enzymatic method (DelaCourt et al., 2022). In this method, sialidase and PNGase F or Endo F3 were used concurrently during the enzyme incubation for sialic acids to be removed from the glycans in addition to cleaving the glycans from the glycoproteins in the tissue. This combined enzymatic approach increased the sensitivity of non-sialylated glycans within the tissue, allowing for enhanced detection and improved spatial visualization of the glycans.

Another major limitation of glycan MSI is differentiating between isomers and isobaric species, since most glycan annotations are assigned using accurate mass. Glycans are very structurally complex and one monoisotopic mass can correspond to multiple glycan compositions, and each composition can have multiple isomers. Linkage information is of particular importance since some pathogens only bind to certain linkages. While branching topology is one of the main interests for linkage determination, sialic acid linkages are extremely important and relevant when it comes to disease. The most common type of sialic acid linkages are α2,3 and α2,6, but they can also form α2,8 and α2,9 linkages. Chemical amidation is the most common type of sialic acid labeling to distinguish α2,3 and α2,6 linkages by accurate mass (West et al., 2021; Zhang et al., 2022). However, there was a recent study that introduced biorthogonal click chemistry to sialic acid labeling by adding alkyne or azide groups to α2,3 and α2,8 linkages (Lu et al., 2023). This was the first time biorthogonal click chemistry was introduced to sialic acid labeling strategies, and provides the foundation for more linkage distinction, especially when pairing this method with ion mobility separations. As mentioned previously, ion mobility can separate isobaric and isomeric species. However, not all mass spectrometers or laboratories have access to these technologies, necessitating the chemical labeling of sugars. Tandem mass spectrometry is another way to elucidate glycan structure. Typical MS/MS on a protonated species produces primarily glycosidic fragments which unfortunately do not provide linkage information. Research has shown that incorporating metal adducts helps stabilize the glycosidic bond and produce cross-ring fragments, which can provide linkage information. Ionization techniques that incorporate an ESI source, such as MALDESI, can incorporate metal-doping to produce cross-ring fragments (Cancilla et al., 1996; Palomino & Muddiman, 2024; Zhou et al., 1990). All of these significant advancements to the field surpass the limits of glycan MSI, allowing for detailed glycomic information to be achieved and further investigated.

8. CONCLUSIONS

MSI technologies and advancements have paved the way to deepen our understanding of glycobiology. Optimization of existing sample preparation techniques have expanded the field to be able to use fresh frozen tissues. Novel ambient ionization methods have successfully detect multiple N-linked glycans without the use of chemical derivatization. Instrumentation and data analysis software continue to advance, and progress has been made in the study of disease progression and pathogenesis. While MSI of N-linked glycans has proven to be a valuable tool in the investigation of disease biology, as with any technique, there remains much to be done. For example, 3D mass spectrometry is rapidly developing and would have substantial effects on the glycomics field (Bai et al., 2020), and sample coverage will expand to include hard tissues such as plants (Zhan et al., 2021). The future of glycan imaging remains promising as it continues to expand to different types of glycans, samples, and methodologies.

ACRONYMS

AD

Alzheimer’s disease

AS

aortic valve stenosis

AUC

area under curve

CE

capillary electrophoresis

CID

collision-induced dissociation

COVID-19

coronavirus disease

DESI

desorption electrospray ionization

EC

endometrial cancer

ECM

extracellular matrix

Endo D

endoglycosidase D

Endo F2

endoglycosidase F2

Endo F3

endoglycosidase F3

Endo H

endoglycosidase H

EOC

epithelial ovarian cancer

ER

endoplasmic reticulum

ESI

electrospray ionization

ETD

electron transfer dissociation

FDR

false discovery rate

FF

fresh frozen

FFPE

formalin-fixed paraffin-embedded

FT-ICR

Fourier transform ion cyclotron resonance

FWHM

full width half max

Gal

galactose

GlcNAc

N-acetylglucosamine

GWB

GlycoWorkbench

HCC

hepatocellular carcinoma

HCD

higher energy collision dissociation

HRAM

high resolution accurate mass

IHC

immunohistochemistry

IR

infrared

IR-MALDESI

infrared matrix-assisted laser desorption electrospray ionization

ITO

indium tin oxide

KOA

knee osteoarthritis

LNM

lymph node metastasis

LPS

lipopolysaccharide

MALDI

matrix-assisted laser desorption ionization

Man

mannose

MASLD

metabolic dysfunction-associated steatotic liver disease

MIRAGE

minimum information required for a glycomics experiment

MMA

mass measurement accuracy

MRI

magnetic resonance imaging

MS/MS

mass spectrometry fragmentation

MS2

mass spectrometry fragmentation

MSI

mass spectrometry imaging

MWCO

molecular weight cut off

Nano-DESI

nanospray desorption electrospray ionization

PD

Parkinson’s disease

PGC-LC

porous graphitized carbon liquid chromatography

PGC-SPE

porous graphitized carbon solid phase extraction

PNGase F

peptide-N-glycosidase F

ppm

parts per million

PSA

polysialic acid

QToF

quadrupole time of flight

RP

resolving power

SARS-CoV-2

severe acute respiratory syndrome coronavirus

S/N

signal-to-noise ratio

SNFG

symbol nomenclature for glycans

TMA

tissue microarray

TOF

time of flight

TW-IMS

traveling wave ion mobility separation

UV

ultraviolet

AUTHOR BIOGRAPHIES

Tana V. Palomino received her Bachelor of Science degree in Chemistry from the College of William & Mary in Williamsburg, V.A. (2020). She is currently a third-year graduate student pursuing a Ph.D. in Chemistry at North Carolina State University. Her research primarily involves investigating sialylation in diseased tissues using mass spectrometry imaging.

David C. Muddiman earned a Bachelor of Science in Chemistry from Gannon University in 1990, and then completed his Ph.D. in Chemistry at the University of Pittsburgh in 1995 under David M. Hercules. He took a postdoctoral appointment at Pacific Northwest National Laboratory between 1995–1997, and his first faculty position was at Virginia Commonwealth University (1997–2002). He was a professor of Biochemistry and Molecular Biophysics at the Mayo Clinic College of Medicine (2000–2002) before beginning his career at North Carolina State University (2005-present) where he is currently a Jacob and Betty Belin Distinguished Professor of Chemistry.