Mitochondrial Preparation from Microglia for Glycan Analysis

Broadly, our research focuses on determining the mechanistic role of sugars, or glycans, and how we can leverage those cellular and subcellular glycosylation pathways in aging as well as brain disorders. Currently, there is a gap in the knowledge about the role as well as modulation of subcellular glycans in different disease pathophysiologies, including the acute and chronic brain disorders like stroke and Alzheimer's. This protocol and our research aims at advancing the knowledge about the role of glycans in neuroimmune interactions, and how that information can be leveraged to design better and efficient central nervous system therapies.

[Narrator] To begin, obtain BV-2 microglial cells derived from C57BL/6 mice. Maintain them in DMEM low glucose medium supplemented with 10% FBS, 1% penicillin streptomycin, and 1% non-essential amino acids. Grow the cells in T175 flasks until they reach 70 to 80% confluency. Aspirate the media from the flask. Resuspend the cell pellet in one milliliter of growth medium. Using trypan blue, count the cells. Centrifuge the cells in a two milliliter microcentrifuge tube at 500 G for five minutes. Carefully aspirate and discard the supernatant. Add 800 microliters of mitochondrial isolation reagent A and vortex at medium speed for five seconds. Then incubate the tube on ice for exactly two minutes. Add 10 microliters of mitochondrial isolation reagent B and vortex at maximum speed for five seconds. Incubate on ice for five minutes, vortexing at maximum speed every minute. Now add 800 microliters of mitochondrial isolation reagent C and invert the tube to mix. And centrifuge at 700 G for 10 minutes at four degrees Celsius. Transfer the supernatant to a new two milliliter tube and centrifuge at 3,000 G for 15 minutes at four degrees Celsius. Transfer the supernatant containing the cytosolic portion to a new tube. The pellet contains the isolated mitochondria. Add 500 microliters of mitochondrial isolation reagent C to the pellet, and centrifuge at 12,000 G for five minutes. Resuspend the isolated mitochondria in 50 microliters of protein isolation buffer. Leave the suspension on ice for 20 minutes. Aspirate and dispense three times, and leave on ice for 20 minutes, vortexing before use. If not fully solubilized, add another 50 microliters of buffer and pool in the same tube. Centrifuge at 13,000 G for 10 minutes. After recovering and freezing the supernatant, dry using a vacuum concentrator. For the detection of released glycans, resuspend the dried and linked glycans in 50 microliters of LCMS grade water. Pipette five microliters of resuspended mitochondrial glycans onto a sample spot on a Teflon microwell slide. Ionize and detect N-glycans in negative ionization mode using an electrospray solvent consisting of 60% acetonitrile and one millimolar acetic acid at a flow rate of two microliters per minute and a voltage of 3.2 kilovolts. Couple IR-MALDESI to a HRAM mass spectrometer set at a resolving power of 240,000 full width at half-maximum at mass-to-charge ratio 200 to analyze between 502,000 mass-to-charge ratio in negative ionization mode. Manually identify the N-linked glycans by searching for monoisotopic masses. Confirm isotopic distributions using mass-to-charge spacing to determine doubly and triply-charged ions with a minimum ion flux threshold of 1,000 ions per second. Convert the raw mass spectra for mass-to-charge ratios to neutral monoisotopic masses. Then upload the monoisotopic masses to an online oligosaccharide structure prediction tool to determine potential glycan compositions. Confirm annotations using an experimentally-curated glyco database. Ensure each identification is within 2.5 parts per million mass measurement accuracy margin, contains the core and linked glycan structure, and excludes pentose, 3-deoxy-d-manno-oct-2-ulosonic acid, or uronic acid monosaccharides. Mitochondrial protein concentrations obtained from six independent preparations showed no significant variation, confirming high reproducibility. Western blood analysis showed CoxIV expression only in the mitochondrial fractions and GAPDH only in cytoplasmic fractions, confirming the purity of mitochondrial isolations and the absence of non-mitochondrial contamination. Distinct sialylated, phosphorylated, and sulfated N-glycan structures were detected in mitochondrial extracts using IR-MALDESI. High squared values testing a goodness of fit confirmed the detection of N-linked glycans with one and two chlorine adducts, confirming the detection of these glycan compositions using IR-MALDESI.