mitochondrial-preparation-from-microglia-for-glycan-analysis

Mitochondrial Preparation from Microglia for Glycan Analysis

Understanding the glycosylation patterns of mitochondrial proteins in microglia is critical for determining their role in neurodegenerative diseases. ...

North Carolina State University

Sialic acids play several roles in both physiological and pathological processes; however, due to their labile nature, they are difficult to analyze using mass spectrometry. Previous work has shown that infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is able to detect intact sialylated -linked glycans without the use of chemical derivatization. In this work, we describe a new rule that can predict the number of sialic acids on a glycan. Formalin-fixed paraffin-embedded human kidney tissue was prepared using previously established methods and analyzed using IR-MALDESI in negative-ion mode mass spectrometry. Using the experimental isotopic distribution of a detected glycan, we can predict the number of sialic acids on the glycan; #sialic acids is equal to the charge state minus the number of chlorine adducts, or  - #Cl. This new rule grants confident glycan annotations and compositions beyond accurate mass measurements, thereby further improving the capability of IR-MALDESI to study sialylated -linked glycans within biological tissues.

Predicting Sialic Acid Content of -Linked Glycans Using the Isotopic Pattern of Chlorine.

-linked glycosylation represents a structurally diverse, complex, co- and posttranslational protein modification that bridges metabolism and cellular signaling. Consequently, aberrant protein glycosylation is a hallmark of most pathological scenarios. Due to their complex nature and non-template-driven synthesis, the analysis of glycans is faced with several challenges, underlining the need for new and improved analytical technologies. Spatial profiling of -glycans through direct imaging on tissue sections reveals the regio-specific and/or disease pathology correlating tissue -glycans that serve as a disease glycoprint for diagnosis. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft hybrid ionization technique that has been used for diverse mass spectrometry imaging (MSI) applications. Here, we report the first spatial analysis of the brain -linked glycans by IR-MALDESI MSI, leading to a significant increase in the detection of the brain -sialoglycans. A formalin-fixed paraffin-embedded mouse brain tissue was analyzed in negative ionization mode after tissue washing, antigen retrieval, and pneumatic application of PNGase F for enzymatic digestion of -linked glycans. We report a comparative analysis of section thickness on the -glycan detection using IR-MALDESI. One hundred thirty-six unique -linked glycans were confidently identified in the brain tissue (with an additional 132 unique -glycans, not reported in GlyConnect), where more than 50% contained sialic acid residues, which is approximately 3-fold higher than the previous reports. This work demonstrates the first application of IR-MALDESI in -linked glycan imaging of the brain tissue, leading to a 2.5-fold increase in the in situ total brain -glycan detection compared to the current gold standard of positive-mode matrix-assisted laser desorption/ionization mass spectrometry imaging. This is also the first report of the application of the MSI toward the identification of sulfoglycans in the rodent brain. Overall, IR-MALDESI-MSI presents a sensitive glycan detection platform to identify tissue-specific and/or disease-specific glycosignature in the brain while preserving the sialoglycans without any chemical derivatization.

Enhanced Detection of Charged -Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging.

Glycans are complex structures that require MS/MS for detailed structural elucidation. Incorporating metals can provide more structural information by inhibiting glycosidic cleavage and enhancing cross-ring fragmentation. A direct analysis was performed using lithium doping and IR-MALDESI to induce cross-ring fragmentation of glycans. The protonated and lithiated versions of the two glycans were isolated and subjected to HCD. For protonated glycans, only glycosidic cleavages were observed. Using lithium doping, MS/MS consisted of abundant cross-ring fragments. Seventeen cross-ring fragments were detected across both glycans using lithium-doped ESI. This is the first incorporation of metal doping in IR-MALDESI to achieve cross-ring fragments in MS/MS analysis.

Achieving Cross-Ring Fragmentation of -Linked Glycans by IR-MALDESI.

With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate 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.

Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances.

Fetal fibrinogen is a variant present in neonates. Blood products used in neonates are tailored for adults and do not seamlessly integrate into neonatal clots. Increased sialic acid content has been found in fetal fibrinogen compared with adult fibrinogen. However, the extent or location of sialic acids on fibrinogen remains unknown.

In-depth characterization of -glycosylation and sialic acid content in fetal and adult fibrinogen.

Chondroitin sulfate (CS) is a type of glycosaminoglycan (GAG) that is abundant in cartilage and perineural networks (PNNs). Changes in the CS signature of PNNs have been implicated in several neurological diseases. Most CS-GAGs contain labile sulfate groups, which can be lost during ionization events that deposit large amounts of internal energy. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft ionization technique used for mass spectrometry imaging. In this work, we determine the spatial distribution of CS-GAG disaccharides within rodent brain using IR-MALDESI MSI. Non-, mono-, and disulfated disaccharides were detected with various adducts. All disaccharides colocalized to the PNNs, which are most abundant in the cortex and hippocampus regions of the brain. This is the first MSI study to spatially resolve CS-GAG disaccharides within brain, paving the way for IR-MALDESI to measure GAGs in neurological diseases.

Article	Year
Predicting Sialic Acid Content of -Linked Glycans Using the Isotopic Pattern of Chlorine. Journal of the American Society for Mass Spectrometry\| PubMed ID: 37289618	2023
Enhanced Detection of Charged -Glycans in the Brain by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometric Imaging. Analytical chemistry\| PubMed ID: 37427925	2023
Achieving Cross-Ring Fragmentation of -Linked Glycans by IR-MALDESI. Journal of the American Society for Mass Spectrometry\| PubMed ID: 38113534	2024
Mass spectrometry imaging of N-linked glycans: Fundamentals and recent advances. Mass spectrometry reviews\| PubMed ID: 38934211	2024
In-depth characterization of -glycosylation and sialic acid content in fetal and adult fibrinogen. Research and practice in thrombosis and haemostasis\| PubMed ID: 39668886	2024
Glycosaminoglycan Mass Spectrometry Imaging by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization. Journal of the American Society for Mass Spectrometry\| PubMed ID: 40033690	2025

Tana V. Palomino

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