Mitochondrial Preparation from Microglia for Glycan Analysis

Meghana Madabhushi; Tana V. Palomino; Mallikarjun H. Patil; David C. Muddiman; Daniel J. Tyrrell; Juhi Samal

doi:10.3791/68179

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

Mitochondrial Preparation from Microglia for Glycan Analysis

用于聚糖分析的小胶质细胞线粒体制备

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06:40 min

May 30, 2025

DOI: 10.3791/68179-v

Meghana Madabhushi¹, Tana V. Palomino², Mallikarjun H. Patil³, David C. Muddiman², Daniel J. Tyrrell³, Juhi Samal⁴

¹School of Health Professions,University of Alabama at Birmingham, ²Department of Chemistry,North Carolina State University, ³Department of Pathology, Division of Molecular and Cellular Pathology,University of Alabama at Birmingham, ⁴Department of Biomedical Engineering,University of Alabama at Birmingham

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Overview

This study presents a protocol for isolating purified mitochondria from microglial cells and detecting subcellular glycans using advanced mass spectrometry techniques. The research aims to enhance understanding of glycan roles in neuroimmune interactions and their implications in brain disorders.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Mass Spectrometry

Background

Glycans play crucial roles in cellular processes and disease mechanisms.
There is limited knowledge about subcellular glycans in brain disorders.
This research addresses the gap in understanding glycan modulation in neuroimmune interactions.
Focus on aging and brain disorders like stroke and Alzheimer's.

Purpose of Study

To develop a protocol for mitochondrial isolation from microglial cells.
To investigate the role of glycans in neuroimmune interactions.
To provide insights for designing better CNS therapies.

Methods Used

Isolation of mitochondria from BV-2 microglial cells using specific reagents.
Detection of N-glycans using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry.
Analysis of glycan structures and compositions using mass spectrometry data.
Validation of mitochondrial purity through Western blot analysis.

Main Results

Successful isolation of mitochondria with confirmed purity.
Detection of distinct N-glycan structures in mitochondrial extracts.
High reproducibility in mitochondrial protein concentrations across preparations.
Identification of glycan compositions using an online prediction tool.

Conclusions

The developed protocol is effective for mitochondrial isolation and glycan analysis.
Findings contribute to understanding the role of glycans in brain disorders.
The research supports potential advancements in CNS therapy design.

Frequently Asked Questions

What are the main applications of this protocol?

This protocol can be used to study mitochondrial functions and glycan roles in neuroimmune interactions.

How does this research impact our understanding of brain disorders?

It provides insights into the modulation of glycans in diseases like Alzheimer's and stroke.

What techniques are used for glycan detection?

Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry is employed.

What cell type is used for mitochondrial isolation?

BV-2 microglial cells derived from C57BL/6 mice are used in this study.

What is the significance of glycans in neuroimmune interactions?

Glycans play a critical role in cellular communication and immune responses in the brain.

How is the purity of mitochondrial isolations confirmed?

Purity is confirmed through Western blot analysis showing specific protein expressions.

开发了一种方案，用于从小胶质细胞制备纯化的线粒体，分离线粒体蛋白以释放 N-糖，以及使用红外基质辅助激光解吸电喷雾电离与高分辨率精确质量分析仪质谱联用快速检测亚细胞线粒体聚糖。

从广义上讲，我们的研究重点是确定糖或聚糖的机制作用，以及我们如何利用这些细胞和亚细胞糖基化途径在衰老和脑部疾病中。目前，关于亚细胞聚糖在不同疾病病理生理学中的作用和调节的知识存在差距，包括中风和阿尔茨海默氏症等急性和慢性脑部疾病。该协议和我们的研究旨在增广有关聚糖在神经免疫相互作用中的作用的知识，以及如何利用这些信息来设计更好、更有效的中枢神经系统疗法。

[旁白]首先，获得源自 C57BL/6 小鼠的 BV-2 小胶质细胞。将它们维持在补充有 10% FBS、1% 青霉素链霉素和 1% 非必需氨基酸的 DMEM 低葡萄糖培养基中。在 T175 烧瓶中培养细胞，直到它们达到 70% 至 80% 的汇合度。从烧瓶中吸出培养基。将细胞沉淀重悬于一毫升生长培养基中。使用台盼蓝，计数细胞。将细胞在两毫升微量离心管中以500G离心五分钟。小心地吸出并丢弃上清液。加入800微升线粒体分离试剂A，以中速涡旋5秒。然后将试管在冰上孵育整整两分钟。加入10微升线粒体分离试剂B，以最大速度涡旋5秒钟。在冰上孵育五分钟，每分钟以最大速度涡旋。现在加入 800 微升线粒体分离试剂 C 并倒置试管进行混合。并在4摄氏度下以700G离心10分钟。将上清液转移到新的两毫升管中，并在 4 摄氏度下以 3,000 G 离心 15 分钟。将含有胞质部分的上清液转移到新管中。该沉淀含有分离的线粒体。向沉淀中加入500微升线粒体分离试剂C，并以12,000G离心5分钟。将分离的线粒体重悬于 50 微升蛋白质分离缓冲液中。将悬浮液放在冰上 20 分钟。吸出并分配 3 次，在冰上放置 20 分钟，使用前涡旋。如果未完全溶解，请再添加 50 微升缓冲液并汇集在同一管中。以 13,000 G 离心 10 分钟。回收并冷冻上清液后，使用真空浓缩器干燥。为了检测释放的聚糖，将干燥和连接的聚糖重悬于 50 微升 LCMS 级水中。将 5 微升重悬的线粒体聚糖移液到聚四氟乙烯微孔载玻片上的样品点上。使用由 60% 乙腈和 1 毫摩尔乙酸组成的电喷雾溶剂，流速为每分钟 2 微升，电压为 3.2 千伏，在负电离模式下电离和检测 N-聚糖。将 IR-MALDESI 耦合到分辨率为 240,000 全宽的 HRAM 质谱仪，质荷比为 200，以半最大分辨率，在负电离模式下分析质荷比为 502,000 之间。通过搜索单同位素质量数手动鉴定 N 连接聚糖。使用质荷间距确认同位素分布，以确定双电荷和三电荷离子，最小离子通量阈值为每秒 1,000 个离子。将质荷比的原始质谱转换为中性单同位素质量。然后将单同位素质量上传到在线寡糖结构预测工具，以确定潜在的聚糖组成。使用实验整理的糖数据库确认注释。确保每次鉴定在百万分之 2.5 质量测量精度裕度范围内，包含核心和连接的聚糖结构，不包括戊糖、3-脱氧-d-甘露-辛基-2-乌洛逊酸或尿酸单糖。从六种独立制剂中获得的线粒体蛋白浓度没有显着变化，证实了高重现性。Western 血液分析显示 CoxIV 仅在线粒体组分中表达，GAPDH 仅在线粒体组分中表达，证实了线粒体分离的纯度和不存在非线粒体污染。使用IR-MALDESI在线粒体提取物中检测到不同的唾液酸化、磷酸化和硫酸化N-聚糖结构。测试拟合优度的高平方值证实了检测到具有一个和两个氯加合物的N-连接聚糖，证实了使用IR-MALDESI检测这些聚糖组成。

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