Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Arun Upadhyay; Robert J. Vassar; Jeffrey N. Savas

doi:10.3791/63816

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

来自大脑的淀粉样原纤维核心的生化纯化和蛋白质组学表征

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09:00 min

April 28, 2022

DOI: 10.3791/63816-v

Arun Upadhyay¹, Robert J. Vassar¹, Jeffrey N. Savas¹

¹Department of Neurology,Northwestern University Feinberg School of Medicine

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Overview

This study presents a biochemical purification method combined with mass spectrometry-based proteomic analysis for the characterization of amyloid fibril cores. The method aims to enhance our understanding of amyloid plaques, which are crucial for the diagnosis and treatment of Alzheimer's disease, by isolating amyloid fibrils at high purity.

Key Study Components

Area of Science

Neuroscience
Biochemistry
Proteomics

Background

Amyloid plaques are essential for the diagnosis of Alzheimer’s disease but not solely sufficient.
Challenges exist in characterizing the contents of amyloid plaques.
Understanding the structure and composition of these plaques may lead to therapeutic interventions.

Purpose of Study

To develop a robust method for purifying and characterizing amyloid fibrils.
To identify potential protein targets for therapies aimed at delaying or preventing Alzheimer's disease onset.
To facilitate exploration of other protein pathways affected in Alzheimer's.

Methods Used

The main platform involves biochemical purification methods combined with mass spectrometry.
Freshly dissected or snap frozen brain tissues are utilized for isolating amyloid fibrils.
The protocol involves multiple centrifugation steps and precise buffer concentrations for effective solubilization and purification.
It includes specific steps for digestion and chromatographic purification of peptides for further analysis.

Main Results

The biochemical method proves efficient in isolating amyloid fibrils in high purity.
Congo red staining confirms enrichment of purified amyloid fibrils.
The study reveals critical steps that enhance the identification of structural components, facilitating target discovery for therapeutic intervention.

Conclusions

This study demonstrates a significant advancement in the purification of amyloid fibrils, which is vital for understanding Alzheimer’s disease.
The method has potential implications for identifying therapeutic targets and delaying disease onset.
Enhanced characterization of amyloid plaques may provide insights into neuronal mechanisms and disease modeling.

Frequently Asked Questions

What are the advantages of the purification method used?

The method is highly efficient at isolating amyloid fibrils with high purity, facilitating detailed structural analysis necessary for therapeutic interventions.

How is the brain tissue prepared for amyloid purification?

Freshly dissected or snap frozen brain tissue is ground using a bead mill homogenizer and then treated with specific buffers in a multi-step purification process.

What types of data can be obtained from this method?

The method yields highly purified amyloid fibrils, enabling structural characterization and proteomic analysis to identify potential therapeutic targets.

How can the method be adapted for other protein aggregates?

The method is designed to extract protein deposits from degenerated tissues, which may be applicable for other protein aggregation disorders as well.

What are the limitations of this protocol?

While effective, the process requires precise control over buffer concentrations and timing, which may introduce variability if not carefully monitored.

这种基于质谱的蛋白质组学分析的生化纯化方法有助于淀粉样原纤维核心的稳健表征，这可以加速预防阿尔茨海默病的靶标的鉴定。

淀粉样蛋白斑块是诊断阿尔茨海默病所必需的。但是，它们还不够。尽管如此，它们仍然神秘，部分原因是在描述其内容方面存在挑战。

我们的实验方案在分离高纯度淀粉样原纤维方面非常有效，非常适合理解结构和成分的精细细节。了解淀粉样蛋白斑块的蛋白质含量可能有助于确定可能延迟、干扰或阻止其积累的治疗干预靶点。因此，延缓疾病的发作。

该方法非常适合从退化的脑组织和不同其他蛋白质途径中的蛋白质聚集体中提取蛋白质沉积物。首先将新鲜解剖或快速冷冻的脑组织区域放在新鲜制备的冰冷均质缓冲液中含有六至八个陶瓷珠的两毫升管中。然后使用珠磨机均质机以4， 000 rpm的频率研磨组织，两个周期为30秒的脉冲和断开脉冲。

现在将9毫升冰冷均质缓冲液加入到1毫升脑组织匀浆液和15毫升管中，并用实验室蜡膜条密封。为确保牢固的溶解，请保持管子在四摄氏度下旋转过夜。第二天，向组织提取物悬浮液中加入固体蔗糖，终浓度为1.2摩尔。

搅拌均匀，离心机在四摄氏度下离心250， 000×g45分钟。弃去上清液后，将沉淀重悬于含有1.9摩尔蔗糖的两毫升均质缓冲液中，方法是在4摄氏度下以125， 000×g研磨和离心45分钟。离心后，将顶部的白色固体层转移到新鲜的管中，并通过上下抚摸几次来溶解一毫升冰冷洗涤缓冲液。

由于颗粒也富含淀粉样原纤维，因此丢弃水性中间层并将颗粒与顶层结合以获得更高的产量。将组合馏分在 8， 000 x g 下在 4 摄氏度下离心 20 分钟。丢弃上清液后，将沉淀重悬于一毫升冰冷消化缓冲液中，并在室温下在涡旋上孵育三小时。

再次离心样品，然后在一毫升冰冷的Tris缓冲液中洗涤沉淀两次，然后再次离心。第二次洗涤后，通过上下移液将沉淀重悬于一毫升增溶缓冲液中，并在4摄氏度下以200， 000×g快速离心管60分钟。保存沉淀，然后通过向上清液中加入50毫摩尔Tris缓冲液将蔗糖浓度从1.3降低到1摩尔。

再次离心上清液，然后溶解含有0.5%SDS的沉淀和100微升Tris缓冲液。对于淀粉样蛋白纯化，使用超声波在浴超声处理装置中溶解富含淀粉样蛋白的颗粒20个循环，然后立即在4摄氏度下以20， 000 x g 离心材料30分钟。将沉淀重悬于500微升的0.5%SDS Tris缓冲液中，并重复洗涤四次，在最后的离心步骤之后，在200微升超纯水中洗涤沉淀，并在4摄氏度下离心20， 000×g30分钟以除去任何剩余的洗涤剂。

将含有纯化的淀粉样原纤维的最终沉淀溶解在100微升超纯水中。将纯化的100微升淀粉样原纤维溶解在400微升甲醇中并充分涡旋。然后混合100微升氯仿并再次涡旋。

接下来，加入300微升超纯水并彻底涡旋。在室温下以12， 000×g离心两分钟后，小心地除去顶部水层而不干扰含有蛋白质片的界面层，并再次加入相同体积的甲醇。重复离心，弃去上清液，风干沉淀。

对于消化，将沉淀溶解在50微升盐酸胍缓冲液中，并在冰冷水浴中超声处理。然后在室温下彻底涡旋45分钟至1小时，加入50微升的0.2%表面活性剂溶液，再次涡旋60分钟。接下来，加入一微升500毫摩尔TCEP并孵育60分钟。

然后加入两微升500毫摩尔碘乙酰胺，并在黑暗中孵育20分钟。孵育后，用5微升TCEP溶液淬灭碘乙酰胺15分钟。接下来，向管中加入所需体积的50毫摩尔碳酸氢铵溶液，以将胍浓度降低至1.5摩尔。

另外，向管中加入1%的表面活性剂溶液。然后加入胰蛋白酶，将管子在37摄氏度下混合过夜。第二天，将甲酸加入消化肽溶液中，将pH降低至2.0，然后通过加入200微升50%甲醇溶液并在室温下以1500×g旋转两分钟来激活C18离心柱。

接下来，通过加入200微升平衡缓冲液并再次旋转两分钟来平衡C18柱树脂床。平衡后，将酸化的肽溶液上样到C18柱上，并在室温下以1500×g离心两分钟。重新加载通过的流，旋转色谱柱，然后丢弃第二个流过。

然后用洗涤缓冲液洗涤与C18树脂结合的肽两次。通过加入40微升洗脱缓冲液并离心色谱柱来洗脱肽三次。通过在蒸发水溶液在快速真空浓缩器中干燥肽。

干颗粒可以在MS分析前在20摄氏度下保存几周。纯化淀粉样蛋白的刚果红染色记录了淀粉样原纤维与SDS可溶性部分相比的富集。SDS可溶性部分未显示淀粉样蛋白的存在。

纯化的原纤维的结构证实了近乎纯的淀粉样原纤维的存在。此外，免疫金标记证实了淀粉样蛋白β-42肽的存在。使用抗淀粉样蛋白β-42和抗原纤维抗体染色显示原纤维中淀粉样蛋白β-42肽相对丰度。

纯化的淀粉样蛋白级分显示存在约250种蛋白质，而超声和SDS洗涤前收集的馏分含有超过2， 500种蛋白质。原纤维核显示与非膜结合细胞器和超分子复合物相关的蛋白质富集。淀粉样蛋白是低丰度物种，可能每毫克脑组织几皮克到几微克。

因此，在拾取层，托盘或丢弃上清液时，您需要非常小心。随着这项技术的发展，您现在可以更自信地鉴定与初始淀粉样蛋白种子相关的蛋白质，表征其结构并靶向它们进行治疗干预。因此，预防这种致命疾病的发作。

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