Name: biochemical purification and proteomic characterization of amyloid fibril cores from the brain
Uploaded: 2022-04-28T13:50:00
Description: Watch this Scientific Journal Video about Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain at JoVE.com

这项工作得到了美国国立卫生研究院授予R01AG061865给R.J.V.和J.N.S.的支持。作者感谢西北大学的瓦萨和萨瓦斯研究小组成员进行了深思熟虑的讨论。我们也衷心感谢博士。安斯加尔·塞默和拉尔夫·兰根在南加州大学为他们的重要投入。我们感谢Farida Korabova博士在西北大学高级显微镜中心进行样品制备和负染色电子显微镜成像。

该协议涉及使用人类或脊椎动物的脑组织。所有研究都是按照西北大学批准的机构指南进行的。目前的工作流程是使用APP敲入（应用程序NL-G-F/ NL-G-F）小鼠大脑皮质和海马脑区域提取物11标准化的。该方案已针对6-9个月大小鼠的脑提取物进行了优化，并且可以有效地纯化雄性和雌性动物的淀粉样蛋白。
注：为了更好地了解整个实验过程，请参?...

<ol>
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对于结构生物学家和生物化学家来说，对淀粉样蛋白结构和组成有清晰的认识是具有挑战性的，因为从AD脑组织中提取纯化的原纤维具有生物学复杂性和实验局限性16，17。淀粉样原纤维在分子水平上是多态的，显示出不同长度和复杂性的异质种群18，19。为了更好地理解它们的生物学特征和病理相关性，需要?...

淀粉样蛋白是一种非常稳定的超分子排列，存在于多种蛋白质组中，其中一些导致病理变化1。在几种神经退行性疾病中观察到细胞内或细胞外淀粉样蛋白聚集体的积累2。淀粉样蛋白聚集体是异质的，富含大量的蛋白质和脂质3。近年来，对淀粉样蛋白组的兴趣引起了基础和转化神经科学家的极大兴趣。已经开发出几种方法从小鼠和死后人类脑组织中提取和纯化淀粉样蛋白聚集体。激光捕获显微切割、免疫沉淀、脱细胞化和淀粉样聚集体的生化分离是提取和纯化淀粉样蛋白斑块、原纤维和低聚物4、5、6、7的广泛使用方法。其中许多研究都集中在使用半定量MS确定这些紧密堆积的纤维沉积物的蛋白质组成。然而，可用的结果是不一致的，并且先前报道的大量共纯化蛋白令人惊讶地难以解释。
描述AD和AD小鼠模型大脑中淀粉样蛋白核心蛋白质组的现有文献的主要局限性在于纯化材料含有难以管理数量的共纯化蛋白。该方法的总体目标是克服这一局限性，并开发一种用于分离淀粉样原纤维核的强大生化纯化方法。该策略采用先前描述的基于超速离心的蔗糖密度梯度超速离心的生化方法，用于从死后AD人和小鼠脑组织中分离SDS不溶性富集淀粉样蛋白级分8，9。该方法建立在现有文献的基础上，但进一步采用超声和SDS洗涤以除去大多数松散结合的淀粉样蛋白相关蛋白，从而分离出高度纯化的淀粉样原纤维（图1）。通过该协议纯化的原纤维克服了从脑提取物中分离的淀粉样原纤维的结构研究中经常遇到的几个现有挑战。用透射电子显微镜（TEM）对这些原纤维进行可视化，确认纯化材料的完整性和纯度（图2）。在这项研究中，分离的原纤维被胰蛋白酶溶解并消化成肽，无标记的MS分析可以很容易地揭示形成原纤维核心的蛋白质的身份。值得注意的是，其中一些蛋白质具有在非膜结合细胞器中形成超分子组件的固有倾向。此外，在淀粉样蛋白β（Aβ）原纤维分析中鉴定的许多蛋白质也与其他神经退行性疾病有关，这表明这些蛋白质可能在多种蛋白质病症中起关键作用。
这种SDS/超声方法不太可能改变或破坏原纤维核的结构。纯化后的材料还适用于各种自上而下和自下而上的蛋白质组学分析方法以及其他基于MS的结构分析策略，例如化学交联或氢氘交换。使用这种方法的总回收率相对较高，因此，适用于详细的结构研究，这需要微克到毫克的纯化材料。纯化后的材料也适用于使用冷冻电镜和原子力显微镜进行结构研究。该协议与哺乳动物的稳定同位素标记相结合，可以促进淀粉样蛋白结构10的固态核磁共振（NMR）研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acclaim PepMap 100 C18 HPLC column 0.075 mm x 20&#160;mm</td>
			<td>Thermo Scientific</td>
			<td>164535</td>
			<td rowspan="35">Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (1-16) 6E10 antibody</td>
			<td>Biolegend</td>
			<td>803001</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (17-24) 4G8 antibody</td>
			<td>Biolegend</td>
			<td>800701</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (N terminus 82E1) antibody</td>
			<td>IBL America</td>
			<td>10323</td>
		</tr>
		<tr>
			<td>anti-amyloid fibril LOC antibody</td>
			<td>&#160;EMD Millipore</td>
			<td>AB2287</td>
		</tr>
		<tr>
			<td>BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
		</tr>
		<tr>
			<td>Bioruptor Pico Plus</td>
			<td>Diagenode</td>
			<td>B01020001</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C1016</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C0130</td>
		</tr>
		<tr>
			<td>Complete&#160; Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0521</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
		</tr>
		<tr>
			<td>HyperSep C18 Cartridges</td>
			<td>Thermo Fisher Scientific</td>
			<td>60108-302</td>
		</tr>
		<tr>
			<td>Integrated Proteomics Pipeline - IP2&#160;</td>
			<td>http://www.integratedproteomics.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
		</tr>
		<tr>
			<td>K54 Tissue Homogenizing System Motor</td>
			<td>Cole Parmer</td>
			<td>Glas-Col 099C</td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://www.maxquant.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23235</td>
		</tr>
		<tr>
			<td>Nanoviper 75 &#956;m x 50&#160;cm</td>
			<td>Thermo Scientific</td>
			<td>164942</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>BR-8101P-E</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion TribridMass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMBCX</td>
		</tr>
		<tr>
			<td>Pierce C18 Spin Columns</td>
			<td>Thermo Fisher Scientific</td>
			<td>89870</td>
		</tr>
		<tr>
			<td>Precellys 24 tissue homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
		</tr>
		<tr>
			<td>ProteaseMAX(TM) Surfactant Trypsin Enhancer</td>
			<td>Promega</td>
			<td>V2072</td>
		</tr>
		<tr>
			<td>RawConverter</td>
			<td>http://www.fields.scripps.edu/rawconv/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>VWR</td>
			<td>97064-646</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>74255</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002446</td>
		</tr>
		<tr>
			<td>Speed Vaccum Concentrator</td>
			<td>Labconco</td>
			<td>7315021</td>
		</tr>
		<tr>
			<td>Tris-2-carboxyethylphosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>15568025</td>
		</tr>
		<tr>
			<td>Trypsin Gold-Mass spec grade</td>
			<td>Promega</td>
			<td>V5280</td>
		</tr>
		<tr>
			<td>UltiMate 3000 RSLCnano System</td>
			<td>Thermo Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
		</tr>
	</tbody>
</table>

biochemical purification and proteomic characterization of amyloid fibril cores from the brain

蛋白纤维包涵体是多种神经退行性疾病的关键病理标志。在阿尔茨海默病（AD）的早期阶段，淀粉样蛋白-β肽在细胞外空间中形成原原原纤维，其作为种子逐渐生长并成熟为大淀粉样蛋白斑块。尽管有这种基本的了解，但目前对大脑中淀粉样原纤维结构，组成和沉积模式的了解是有限的。一个主要的障碍是无法从脑提取物中分离出高度纯化的淀粉样原纤维。基于亲和纯化和激光捕获显微切割的方法以前曾用于分离淀粉样蛋白，但受到可回收少量材料的限制。这种新颖、稳健的方案描述了使用十二烷基硫酸钠（SDS）增溶与蔗糖密度梯度超速离心和超声处理对淀粉样蛋白斑块核心的生化纯化，并从AD患者和AD模型脑组织中产生高纯度的原纤维。基于质谱（MS）的纯化材料自下而上的蛋白质组学分析代表了一种强大的策略，可以识别淀粉样原纤维的几乎所有主要蛋白质成分。以前对淀粉样蛋白冠状动脉中蛋白质的蛋白质组学研究揭示了出乎意料的大和功能多样化的蛋白质集合。值得注意的是，在提纯策略后，共纯化蛋白的数量减少了10倍以上，表明分离出的SDS不溶性物质纯度高。阴性染色和免疫金电子显微镜检查可以确认这些制剂的纯度。需要进一步的研究来了解导致这些蛋白质沉积成淀粉样蛋白内含物的空间和生物学属性。总而言之，这种分析策略可以很好地增加对淀粉样蛋白生物学的理解。

这里，总结了使用修饰蔗糖密度梯度超速离心纯化方法分离和纯化淀粉样原纤维的详细方法（见 图1）。该方法的创新之处在于包括使用水浴超声处理系统进行基于超声波的洗涤步骤，然后进行SDS增溶，从淀粉样原纤维中除去许多松散相关的蛋白质，这些蛋白质与高度致密和干净的原纤维共纯化。超声步骤产生高剪切力并搅拌原纤维，松动疏水力并将SDS可溶性松散相关的蛋?...

Watch this Scientific Journal Video about Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain at JoVE.com

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

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。

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

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

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。

Research

JoVE Journal

Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Selection of Aptamers for Amyloid ÃƒÅ½Ã‚Â²-Protein, the Causative Agent of Alzheimer's Disease

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

科研

神经科学

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Purification of Mouse Brain Vessels

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Visualization of Amyloid ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â² Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

矩阵辅助激光去离子成像质谱法显示人脑中的淀粉样β矿床

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Purification of the Dendritic Filopodia-rich Fraction

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...