Mitochondrial Preparation from Microglia for Glycan Analysis

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.

Results

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 BC...

Discussion

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 identifi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Amersham 600 imager	Cytvia	29194217	Gel and membrane imager
Countess 3 automated cell counter	Fisher Scientific	X003SZ1LY9
Dry Bath Stdrd 4 blck 100-120V	Thermofisher scientific	88870003
i-Blot2 Gel Transfer Device	Invitrogen	IB21001	Western blot transfer system
Inverted microscope	Cell Treat	04355223EA
Microplate reader		82050-760
Mini gel tank	Invitrogen	A25977
Open Air Rocker	Fisher Brand	88861025
Pipet boy	BioTek	229310
Vortex mixer	Integra- VWR
Mitochondria isolation reagents
Mitochondrial Isolation kit	Thermofisher scientific	89874
Phosphotase Inhibitor	Thermofisher scientific	1861274
Protease Inhibitor	Thermofisher scientific	1861281
N-glycan isolation and IR-MALDESI reagents
Acetic acid	Fisher Scientific	A11350	50% in ESI solvent
Acetonitrile	Sigma Aldrich	34851-4L	1 mM in ESI solvent
Ammonium bicarbonate	Fisher Scientific	A643500	100 mM
Calibration Solution	Thermofisher Scientific	A39239	Pierce FlexMix
Dithiothreitol	Sigma Aldrich	AC426380100	1 M
Iodoacetamide	Sigma Aldrich	A322-10VL
LC/MS grade water	Thermofisher Scientific	047146.M6
PNGase F	Bulldog Bio	NZPP010	75000 U/mL, enzyme for N-glycan release
N-glycan isolation and IR-MALDESI consumables
Amicon centrifugal filters	Fisher Scientific	UFC501024	10 kDa MWCO
Mass spectrometer	Orbitrap Exploris 240
Mid-IR Laser	JGM Associates, Burlington, MA, USA
Teflon microwell slide	Prosolia, Indianapolis, IN, USA
N-glycan analysis softwares
GlycoMod	Expasy		https://web.expasy.org/glycomod/
GlyConnect	Expasy		https://glyconnect.expasy.org/
Protein isolation and western blot consumables
Basix gel loading tips ( 10 µL)	Basix	13-611-102
Basix gel loading tips ( 200 µL)	Basix	13-611-116
Cell scrapper	VWR labs	14-388-100
i-Blot NC regular stacks	Invitrogen	IB23001
i-Blot2 PVDF Regular Stacks	Invitrogen	IB24001
10  µL micropipette	Fisher Scientific	FBE00010
20 µL micropipette	Invitrogen	FBE00020
200  µL micropipette	Fisher Brand	FBE00200
1000 µL micropipette	Fisher brand	FBE01000
10  µL pipet tips	VWR labs	76322-528
20  µL pipet tips	VWR labs	76322-134
200  µL pipet tips	VWR labs	76322-150
1000  µL pipet tips	VWR labs	76322-154
Well plate	Fisher brand	14-388-100
Protein isolation and western blot reagents
Actin antibody ( Host : Rabbit )	Cell Signaling Technologies	8457T
Anti-Rabbit IgG HRP Linked	Cell Signaling Technologies	7074S
Bolt 4-12% Bis-Tris Plus	Invitrogen	NW04120BOX
Bovine Serum Albumin	Fisher bioreagents	BP9700-100
COXIV antibody ( Host : Rabbit)	Cell Signaling Technologies	4844S
GAPDH antibody ( Host : Rabbit)	Cell Signaling Technologies	2118S
MicroBCA protein assay Kit	Thermofisher scientific	23235
Nupage MOPS SDS Runing Buffer [20x]	Thermofisher scientific	NP0001
PAGE Ruler prestained protein ladder	Thermofisher scientific	815-968-0747	Dilution= Use 7  µL to load onto first well
Phosphate buffered saline	Aniara Diagnostics	A12-9423-5	Prepare 1x PBS from 10x powder
Pierce ECL Western Blotting Substrate	Thermofisher scientific	32106	Chemiluminescent substrate kit
RIPA Buffer	Thermofisher scientific	89901
Sample Buffer	Novex	B0007	The bolt LDS sample buffer is prepared in 3:1 ratio of sample to sample buffer
Tween-20	MP Biomedicals	TWEEN201
Tissue culture consumables
Countess Slides	Avantor	229411
Eppendorf tubes	Cell Treat	414004-265612-5884
2 mL aspirating pipet	Vista lab	5090-0010E
5 mL serological pipet	Fisher Scientific	13-678-11D
10 mL serological pipet	Basix	13-678-11E
25 mL serological pipet	Vista lab	FB012937
50 mL serological pipet	Vista lab	14955233
15 mL Conical tube	Avantor	229225A
50 mL conical tube	Cell treat	4190-0050
T-75 cm2 Tissue culture flask	Fisher Scientific	FB012937
T-180 cm2 Tissue culture flask	Fisher Scientific	FB012939
Tissue culture reagents
BV2 microglial cell line	Creative Bioarray	CSC-I2227Z	Immortalized Mouse Microglia (BV2) derived from C57/BL6 neonatal microglia
Cell dissociation enzymes	Thermofisher scientific	12563029	TrypLE
Dulbecco's Modified Eagle Medium (DMEM) Low Glucose Media	Gibco	10567014
Fetal Bovine Serum	Cytiva	SH30071.03HI
Minimum Essential Medium (MEM) Non-essential Amino Acids	Gibco	11140050
Penicillium Streptomycin	Cytivia	SV30010
Phosphate buffer saline	Corning	21-040-CV
Trypan Blue stain 0.4%	Invitrogen	T10282