Mitochondrial Preparation from Microglia for Glycan Analysis

A protocol was developed for the preparation of purified mitochondria from microglial cells, isolation of mitochondrial proteins for N-glycan release, and rapid detection of subcellular, mitochondrial glycans using infrared matrix-assisted laser desorption electrospray ionization coupled to high-resolution accurate mass analyzer mass spectrometry.

Understanding the glycosylation patterns of mitochondrial proteins in microglia is critical for determining their role in neurodegenerative diseases. Here, we present a novel and high-throughput methodology for glycomic analysis of mitochondrial proteins isolated from cultured microglia. This method involves the isolation of mitochondria from microglial cultures, quality assessment of mitochondrial samples, followed by an optimized protein extraction to maximize glycan detection, and infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) high-resolution accurate mass (HRAM) mass spectrometry to provide detailed profiles of mitochondrial glycosylation.

This protocol emphasizes the importance of maintaining mitochondrial integrity during isolation and employs stringent quality control to ensure reproducibility, including measuring mitochondrial purity after extraction. This approach allows for the comprehensive profiling of glycosylation changes in microglial mitochondria under various experimental conditions in vitro, which offers insight into mitochondrial changes associated with neurodegenerative diseases. This approach could be adapted to other in vitro treatments, other cultured cell types, or primary cells. Through this standardized approach, we aim to advance the understanding of microglial mitochondrial glycans, contributing to the broader field of neurodegenerative research.

Microglia are the dominant resident innate immune cells in the brain and account for 10-15% of cells in the adult brain^1,2. They use their receptor repertoire to dynamically monitor the brain microenvironment and regulate the normal brain function to maintain brain homeostasis³. Microglia are very sensitive to the changes in their microenvironment and undergo changes in cell morphology, immunophenotype, and function with pathological conditions or various stimulations. Microglial activation states are influenced by the cellular energy demands required for their function, such as phagocytosis, cytokine production, or tissue repair. Therefore, cellular energy metabolism plays a crucial role in regulating changes in microglial function⁴. Microglial dysregulation leads to excessive release of pro-inflammatory cytokines (e.g., IL-1β, TNF-α) and reactive oxygen species (ROS), predisposing the brain to neuroinflammation^5,6. Chronic microglial dysregulation and the resulting neuroinflammatory environment lays a foundation for neurodegeneration⁷.

The brain accounts for only 2% of body weight but 20% of the body's total energy consumption. Mitochondria are the primary source of energy in brain cells and act as key players in the pathogenesis of both acute and chronic brain disorders⁸. Previous studies have established a strong correlation between microglial activation and metabolic dysfunction in aging⁹ and age-related disorders such as Alzheimer's disease^10,11, highlighting the pivotal role of mitochondria in cellular senescence and neurodegeneration. Impaired mitochondrial function leads to diminished energy production, elevated oxidative stress, and increased neuroinflammation during aging and age-related diseases.

While extensive research has elucidated the role of mitochondria in energy metabolism, aging, and brain disorders, the role of common post-translational modifications, such as glycosylation, in mitochondrial biology and function remains insufficiently explored. Glycosylation, the enzymatic addition of sugar moieties called glycans to proteins by glycosylation enzymes, is the most common post-translational modification in most brain cells, including microglia. Activated microglia modulate their immune function under inflammatory stimuli by regulating the intracellular or cell surface glycan expression¹². The pro- and anti-inflammatory responses exhibited by microglia post-stimulation are also regulated by the glycans¹³. Mitochondrial proteins also have these glycan modifications, which regulate their function and localization. However, detailed analysis of the cell-specific mitochondrial glycosylation patterns in the microglia is lacking due to the technical challenges in investigating sub-cellular glycosylation. Despite the well-characterized roles of glycosylation in modulating the microglial phenotype, the role of glycans in modulating mitochondrial function and subsequently, cellular immunophenotype in microglia remains poorly understood.

Limited studies investigating mitochondrial protein glycosylation have focused primarily on lectin-based identification of glycosylation patterns. Lectins are glycan-binding proteins that bind biomolecular glycan moieties^14,15, which lack the specificity and ability to provide detailed information about the glycan composition. Mass spectrometric modalities offer a detailed identification of the glycan compositions to overcome the analytical challenges presented by lectin analysis. One such modality, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), employs a hybrid ionization strategy, using a mid-IR laser to resonantly excite water found in biological specimens¹⁶ to desorb the neutral species and subject them to an orthogonal electrospray plume, followed by analysis using a high-resolution accurate mass Orbitrap mass spectrometer. IR-MALDESI has been previously demonstrated for the direct analysis of tissue metabolites¹⁷, with distinct advantages of rapid analysis¹⁸, soft ionization method, and the predictability of sialic acid content of N-linked glycans based on the isotopic distribution patterns of chlorinated glycan adducts¹⁹. However, the adaptation of this platform for the direct analysis of sub-cellular glycans has not been demonstrated.

Here, we report a high-throughput protocol for mitochondrial isolation from microglial cells, isolation of mitochondrial N-glycans, and mitochondrial N-glycan detection and analysis using IR-MALDESI mass spectrometry. This protocol will be foundational in uncovering novel insights into the role of glycosylation in mitochondrial function, potentially identifying new therapeutic targets for neuroinflammatory and neurodegenerative disorders.

1. BV2 Microglial cell line culture

1. Maintain the BV-2 cells (microglial cells derived from C57BL/6 mice) in DMEM low glucose media supplemented with 10% Fetal Bovine serum (FBS), 1% Penicillium-Streptomycin (PenStrep), and 1% Non-essential Amino Acids (NEAA).
2. Grow the cells in T-175 flasks and allow them to reach 70-80% confluency.
3. Split the cells by incubating with the cell dissociation enzymes for 5 min at 37 °C in 5% CO₂, followed by inactivation of the enzymes with an equal volume of cell media, and centrifugation of cells for 5 min at 500 × g at room temperature (RT).
   NOTE: The cell dissociation enzymes used here are gentler on BV2 cells than trypsin and can be used to protect the cell surface antigen expression.
4. Aspirate the media, resuspend the cell pellet in 1 mL of growth media, and count the cells using trypan blue (see the Table of Materials).
   NOTE: We used an automated cell counter for this step to count cells.
5. To carry-out the mitochondrial isolation, proceed if the cell pellet contains 2 × 10⁷ (20 million cells/pellet).

2. Isolation of mitochondria from microglial cells

NOTE: Work quickly, keeping everything on ice throughout the procedure. The mitochondrial isolation kit used for mitochondrial isolation has three components: Reagents A (cell lysis buffer), Reagent B (stabilizing buffer), and Reagent C (mitochondrial wash buffer). Add protease inhibitors to reagent A and reagent C immediately before use.

1. Pellet 2 × 10⁷ cells by centrifuging harvested cells in a 2.0 mL microcentrifuge tube at 500 × g for 5 min. Carefully aspirate and discard the supernatant.
2. Add 800 µL of mitochondrial isolation reagent A (cell lysis buffer), vortex at medium speed for 5 s, and incubate the tube on ice for exactly 2 min.
   NOTE: Do not exceed the 2 min incubation.
3. Add 10 µL of mitochondrial isolation reagent B (stabilizing buffer), vortex at maximum speed for 5 s, and incubate the tube on ice for 5 min, vortexing at maximum speed every minute.
4. Add 800 µL of mitochondria isolation reagent C (mitochondrial wash buffer), invert the tube several times to mix, and centrifuge the tube at 700 × g for 10 min at 4 °C.
   NOTE: Do not vortex.
5. Transfer the supernatant to a new 2.0 mL tube and spin at 3,000 × g for 15 min at 4 °C.
6. Transfer the supernatant (cytosolic portions) to a new tube. The pellet contains the isolated mitochondria.
7. Add 500 µL of Mitochondria isolation reagent C to the pellet, and centrifuge at 12,000 × g for 5 min.
8. Use the pellet for protein quantification and processing or store the pellet at -80 °C until further use.

3. Protein estimation using microBCA assay

NOTE: Protein estimation for this protocol can be performed using different reagents and assays. Quantification of cytosolic or mitochondrial proteins can be performed by normalizing against the total protein concentration used in the assay.

1. Prepare bovine serum albumin (BSA) standards between 0 µg/mL and 200 µg/mL (0 µg/mL, 0.5 µg/mL, 1 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, 200 µg/mL) and blanks with lysis buffer only.
2. Add 150 µL of each standard into the flat-bottomed 96-well plate containing the samples.
3. Mix 25 parts of Micro BCA Reagent MA (bicinchoninic acid (BCA) solution) and 24 parts Reagent MB (copper sulfate solution) with 1 part of Reagent MC (stabilizing buffer) (25:24:1, Reagents MA:MB:MC) to create a working reagent. Add 150 µL of the mixed BCA reagent to each sample and standard.
4. Incubate at 37 °C for 2 h.
5. Use a plate reader to measure the absorbance at 562 nm and quantify the protein concentration with the standard curve. Subtract the blank standard absorbance from the absorbance measurement of all other individual standards and unknown sample replicates to obtain the sample protein concentrations.

4. Mitochondrial preparation quality control (western blot)

1. Resuspend the mitochondrial pellet in the radioimmunoprecipitation assay buffer (RIPA) buffer to perform protein quantification. Determine the protein concentration for each sample using the micro BCA assay.
2. Denature the samples in sample buffer at 94 ^oC for 5 min.
3. Load equal amounts (20 µg) of mitochondrial protein to precast gels (see Table of Materials), along with the molecular weight marker.
4. Run the gel for 50 min at 100 V.
   NOTE: The running time may vary, so make sure to stop the gel when the proteins have reached the end of the gel indicated by the dye in the sample buffer.
5. Transfer the proteins from the gel to a polyvinylidene fluoride (PVDF) membrane using the dry-transfer protocol in a western blot transfer system for 7 min at 20 V.
6. Take the membrane out and block using the blocking solution (Table 1) for 1 h at room temperature.
7. Add appropriate dilutions of primary antibody to the membrane in blocking buffer overnight at 4 ^oC (COXIV= 1:3,000, GAPDH =1:3,000).
   NOTE: The absence of nuclear, endoplasmic reticulum (ER) contamination in the mitochondrial preparation can be tested by using additional markers like lamin (nuclear marker) and ERp57 (ER marker) in western blots.
8. Wash the membrane for 3 x 15 min with the wash buffer (Table 1).
9. Incubate the membrane with the appropriate dilution of HRP-conjugated secondary antibody in blocking buffer at room temperature for 1 h
10. Wash the membrane for 3 x 15 min with the wash buffer.
11. For developing, use a chemiluminescent substrate kit.
12. Scan the membrane using a gel and membrane imager.

5. Mitochondrial protein isolation for N -glycan extraction from microglia

1. Resuspend the isolated mitochondria in 50 µL of protein isolation buffer (Table 1) and leave on ice for 20 min.
2. Aspirate and dispense three times and leave 20 min on ice (vortex before use). If the mitochondrial pellet is not fully solubilized, add another 50 µL of the isolation buffer and pool into the same tube.
3. Centrifuge at 13,000 × g for 10 min.
4. Recover the supernatant, freeze at -80 ^oC for a minimum of 1 h, and dry as much as possible in a vacuum concentrator.
5. Resuspend before glycan isolation using the PNGase digest buffer (Table 1) for IR-MALDESI.

6. Mitochondrial N -glycan preparation for IR-MALDESI

1. Load 25-250 µg of protein (in 250 µL maximum volume) of isolated mitochondrial proteins onto a 10 kDa molecular weight cut-off (MWCO) filter.
2. Reduce the protein samples to expose the glycan moieties by adding 2 µL of 1 M dithiothreitol (DTT) to each sample in the filter.
3. Dilute the sample with 200 µL of PNGase digest buffer and vortex lightly to avoid disturbing the filter.
4. Denature the mitochondrial proteins by incubating the sample at 56 °C for 30 min.
5. Use 50 µL of 1 M iodoacetamide to alkylate the mitochondria to give a final concentration of ~200 mM, and incubate at 37 °C for 60 min.
6. To further concentrate the denatured mitochondrial methods, centrifuge the samples at 14,000 × g for 40 min. Discard the flowthrough.
7. Wash the sample with 100 µL of PNGase digest buffer.
8. Concentrate the glycoprotein on the filter at 14,000 × g for 20 min and discard the flowthrough. Repeat the wash and concentrate step 2x, for a total of 3x, yielding a concentrate in the filter dead volume (~5 µL). Discard all flowthrough.
9. Discard the collection vial once the washes are complete. Use a new collection vial for all future eluents and washes.
10. To cleave the glycans from denatured mitochondrial glycopatterns, transfer to a fresh collection vial and add 2 µL of glycerol-free PNGase (75,000 units/mL) to the filter. Add 98 µL of PNGase digest buffer, bringing the total volume to 100 µL and mix by gently pipetting up and down on the filter.
11. Incubate samples at 37 °C for 18 h to enzymatically cleave all N-glycans from mitochondrial proteins.
12. Elute the released mitochondrial N-glycans by centrifuging the sample at 14,000 × g for 20 min at 20 °C.
13. Wash the mitochondrial glycans by adding 100 µL of PNGase digest buffer to the filter and centrifuge at 14,000 × g for 20 min at 20 °C. Collect the wash containing any remaining N-glycans in the same collection vial as the eluent. Repeat 2x and remove the filter from the collection vial.
14. Incubate the mitochondrial glycan samples in the -80 °C freezer until frozen (30-60 min) and dry to completion at room temperature in a vacuum concentrator (4-6 h for 400 µL).
    NOTE: N-glycans may be stored at -20 °C for up to 6 months prior to analysis.
15. Resuspend the dried N-linked glycans in 50 µL of LC/MS grade water directly before IR-MALDESI analysis.

7. Detection of released glycans by IR-MALDESI mass spectrometry

1. Perform mass calibration each day of the experiment. Load the calibration solution onto a syringe pump and push through at a rate of 1.2 µL/min. Apply a voltage of 3.5 kV to achieve a stable electrospray plume for mass calibration in both positive and negative modes.
2. Pipette 5 µL of resuspended mitochondrial glycans onto a sample spot on a Teflon microwell slide.
3. Use a mid-IR laser operating at a wavelength of 2.97 µm for ablation with an energy of 1.8 mJ per burst.
4. Ionize and detect N-glycans in negative ionization mode. Use electrospray solvent consisting of 60% acetonitrile and 1 mM acetic acid to create a stable electrospray plume with a flow rate of 2 µL/min at a voltage of 3.2 kV.
5. To perform the analysis, couple IR-MALDESI to a HRAM mass spectrometer set to a mass resolving power of 240,000_FWHM at m/z 200, analyzing between 500 and 2,000 m/z in negative ionization mode.
6. Turn off automatic gain control (AGC) and set a fixed injection time of 90 ms. Use the EasyIC source for real-time internal calibration of every spectrum to achieve high mass measurement accuracy (MMA).

8. Mitochondrial N- glycan data analysis

1. Manually identify the N-linked glycans by searching for monoisotopic masses and confirming isotopic distributions using the m/z spacing to determine doubly and triply charged ions that have a minimum ion flux threshold of 1,000 ions/s.
2. Convert the raw mass spectra from m/z ratios to neutral monoisotopic masses.
3. Upload the monoisotopic masses to an online oligosaccharide structure prediction tool to determine potential glycan compositions. Confirm annotations using an experimentally curated glycomic database²⁰; ensure that each identification is within the margin of 2.5 ppm MMA, contains the core N-linked glycan structure (Hex₃HexNAc₂), and excludes pentose, KDN, or HexA monosaccharides.
4. Draw the confirmed glycan structures using SNFG nomenclature²¹.
5. Obtain the relative abundance of glycans from the raw mass spectra and normalize against the amount of mitochondrial protein in µg to obtain ions/s/µg.
6. Use Chi-squared analysis to test the goodness of fit between the theoretical and experimental distributions of the N-linked glycans to determine the number of chlorine adducts. This allows direct determination of the number of sialic acids¹⁹.

Figure 1 represents a schematic outline of the steps involved in the isolation of mitochondria from the BV2 microglial cell line for mass spectrometric glycan analysis. The reproducibility of mitochondrial protein isolation between different mitochondrial preparations from the same starting density of the microglial cells is represented in Figure 2, which shows no significant difference between the mitochondrial protein concentration estimated using the micro BCA assay.

Figure 3 represents the purity of mitochondrial isolations using western blot analysis of COX IV and GAPDH. Here, we see the expression of the mitochondrial protein COX IV in the mitochondrial fraction isolated in the final step of the reagent-based isolation used in the protocol. The immunoblots show a prominent COX IV band in the isolated mitochondrial fraction, while GAPDH is only detected in the cytoplasmic fraction at the same exposure. Longer exposure times can result in the detection of a faint GAPDH band. Expression of the non-mitochondrial marker GAPDH is evident in the whole cell lysate after isolation of mitochondria, without the CoxIV bands, indicating a complete isolation of the mitochondrial fraction and minimal non-mitochondrial contamination. COX IV expression in the mitochondrial fractions is consistent between different preparations with similar starting cell density of 2 × 10⁷ cells.

Representative mass spectra of the released N-glycans detected using IR-MALDESI in Figure 4 shows the presence of several phosphorylated, sulfated, and sialylated charged N-glycans released from the mitochondrial protein extract using PNGase treatment. Table 2 reports all the glycan compositions that were identified in the whole spectra but were not reported in GlyConnect. Chi-squared values testing a goodness-of-fit confirm the detection of N-linked glycans with one and two chlorine adducts, confirming the detection of these glycan compositions using IR-MALDESI in Figure 5.

Figure 1: Protocol outline. Schematic outline of the mitochondrial isolation, quality control, protein extraction, N-glycan release, and estimation using IR-MALDESI HRAM mass spectrometry from BV2 microglial cell line for high throughput detection of mitochondrial N-glycans. Abbreviation: IR-MALDESI HRAM = Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization High-Resolution Accurate Mass Analyzer. Please click here to view a larger version of this figure.

Figure 2: Mitochondrial protein isolation from BV2 cells. (A) Standard curve for micro BCA assay using different concentrations of BSA. (B) Reproducibility of the mitochondrial protein isolation represented by consistent protein content from 2 × 10⁷ cells in six independent mitochondrial preparations. Please click here to view a larger version of this figure.

Figure 3: Purity of mitochondrial preparation. Representative western blot for cytoplasmic fraction as well as isolated mitochondria from BV2 microglial cells. The figure represents immunoblotting of the cytoplasmic fraction and the isolated mitochondria with COX IV antibody (mitochondrial marker) and GAPDH antibody (cytoplasmic control). The absence of GAPDH bands in the isolated mitochondria indicates minimal cross-contamination and the purity of mitochondrial preparation for subsequent N-glycan analysis. Please click here to view a larger version of this figure.

Figure 4: Mitochondrial N-glycan identification composition determination using IR-MALDESI HRAM mass spectrometry. Glycan mass spectra in the range of 1,700-2,000 m/z showing a significant number of multiply-charged peaks with the annotated structures determined using Glycomod. Abbreviation: IR-MALDESI HRAM = Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization High-Resolution Accurate Mass Analyzer. Please click here to view a larger version of this figure.

Figure 5: Verification of the detected mitochondrial N-glycans. Representative isotopic distributions for four (A-D) mitochondrial N-linked glycans showing an overlay of the observed distribution with theoretical distributions of chlorine and deprotonated adducts. Chi-squared values on the overlaid spectra represent the goodness-of-fit and confirm the detection of N-linked glycans with one and two chlorine adducts. This is further confirmation of the detection of these glycan compositions using IR-MALDESI. Abbreviation: IR-MALDESI = Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization. Please click here to view a larger version of this figure.

Table 1: Solution and buffer recipes used in the protocol. Please click here to download this Table.

Table 2: Multiply charged deprotonated N-linked Glycans detected in the mitochondria with high mass measurement accuracy in Glycomod. Glycan short-hand notation: H = hexose; N = N-acetylglucosamine; F = fucose; S = N-acetylneuraminic acid; Phos = phosphate; Sulph = sulfate modification. Please click here to download this Table.

Microglia are the resident immune cells of the brain, and glycan modifications modulate the immunophenotype and function of microglia. These immune functions demand substantial cellular energy, which is predominantly supplied by mitochondria. Notably, the mitochondrial proteins also present glycan modifications, which have remained significantly understudied due to the technological challenges in investigating sub-cellular glycosylation. Most studies investigating mitochondrial glycosylation rely on lectin-based identification of glycan patterns²², though these approaches are limited by the poor binding specificity of lectins. The essential advances of the technical approach presented in this study are i) reproducible isolation of mitochondrial N-glycans from microglial cells and ii) detection and identification of mitochondrial N-glycans using IR-MALDESI HRAM mass spectrometry. The workflow described here is the first report of the detection of N-glycans released from mitochondrial glycoproteins expressed at physiological levels in microglial cells.

In the present protocol, mitochondrial isolation is maximized by using the reagent-based isolation method. This can be combined with the Dounce homogenization method to improve the mitochondrial yield. A drawback of using the homogenizer compared to the reagent-based isolation is the variation in the force and speed of the pestle between operators, which results in increased experimental variation and reduced reproducibility. Prior studies^23,24 have indicated the use of this method and the absence of nuclear (lamin, histone H3) and ER markers (calnexin, Erp57) in the mitochondrial fraction, indicating the purity of the mitochondrial fraction. A potential limitation of the protocol is the requirement of 20 million cells as a starting material for mitochondria isolation. However, the high mitochondrial protein concentration observed in our study allows for a ten-fold scale down of initial microglial cell number for mitochondrial isolation based on the optimization performed in prior studies^25,26 (25-250 µg proteins) without losing any N-glycan signals. Furthermore, pooling biological samples to obtain higher cell numbers for mitochondrial isolation could be performed in case of limited protocol scalability to lower cell numbers from primary sources like tissues for efficient glycan detection. In addition, the glycan release step is critical in this protocol. N-glycosidase F (PNGase F) is used to release complete and intact N-linked glycans by hydrolyzing the amide link between the innermost N-acetylglucosamine (GlcNAc) and the asparagine residue²⁷. For N-glycan analysis, it is critical to optimize the activity of PNGase F to achieve full de-glycosylation of the mitochondrial proteins. Protein denaturation using solvent exposure and excess enzyme helps ensure efficient and complete cleavage and release of the N-glycans from the mitochondrial proteins. A digestion time of 18-20 h is optimal for N-glycan release by completing the PNGase F reaction²⁶.

A potential limitation of this protocol is the lack of enrichment of the mitochondrial extract for glycoproteins in this method. While the low-abundance glycoproteins may not be detected in the analysis, this method minimizes the error and bias introduced from lectin affinity purification or chemical enrichment. While IR-MALDESI gives high-confidence identification of the composition of the identified glycans, precise information about the linkages between the glycan residues requires further investigation using tandem mass spectrometry of lithium-adducted glycans to enhance cross-ring cleavages²⁸ or exoglycosidase digestions. Alternatively, LC-MS/MS can be used in addition to or as an alternative to the IR-MALDESI approach, which may provide deeper glycan coverage with more structural details. However, previous studies have shown the correlation between the average ion abundance from IR-MALDESI for metabolites with the absolute quantities determined by LC-MS/MS²⁹, establishing a foundation for direct quantitation of metabolites using IR-MALDESI. The high-throughput nature of IR-MALDESI with an ability to perform MS² analysis to provide a high-confidence structural confirmation is instrumental for rapid screening of preclinical and clinical samples for potential glycan biomarkers and disease diagnosis. In addition, it should be noted that although the centrifugation of the post-nuclear supernatant at 3000 × g instead of at 12,000 × g minimizes the peroxisomal and lysosomal contaminants, there may still be traces of these proteins present in the mitochondrial preparation.

In conclusion, this study presents a simple, high-throughput method, with minimal sample preparation for the analysis of the mitochondrial glycome in microglial cells and great potential for application in the identification of novel glycan-based therapeutic targets for neurodegenerative diseases. This protocol presents a comprehensive evaluation and standardization of the analytical methods for mitochondrial isolation from microglia and subsequent analysis of the mitochondrial glycome to enable scale-up for large-scale preclinical and clinical studies. This protocol presents several advantages for N-glycan isolation and detection: i) the mitochondrial isolation time for reagent-based protocol is low (≤40 min); ii) the yield of proteins for glycan release is high; iii) same mitochondrial samples prepared for N-glycan analysis can be used for other molecular biological investigations such as proteomic analysis, lectin analysis and mitochondrial flow analysis; and iv) IR-MALDESI HRAM mass spectrometry allows for rapid and improved detection of N-glycans due to the hybrid and soft ionization strategy²⁸ without the need for chemical derivatization of the charged glycans like sialoglycans and sulfoglycans³⁰.

The authors have no conflicts of interest to declare.

The authors would like to thank Seth Eisenberg, graduate student in Muddiman Lab at NCSU, for his help with video recording of mass spectrometric protocol. This research was supported in part by the School of Engineering Innovation Fellows Program at the University of Alabama at Birmingham, AG068309 to D.J.T. and R01GM087964-12 to D.C.M. The schematics in this manuscript were drawn using BioRender.