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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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?…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Acclaim PepMap 100 C18 HPLC column 0.075 mm x 20 mm | Thermo Scientific | 164535 | Alternative instruments, chemicals and antibodies from other manufacturers can be used |
| Ammonium bicarbonate | Sigma-Aldrich | 9830 | |
| anti-amyloid beta (1-16) 6E10 antibody | Biolegend | 803001 | |
| anti-amyloid beta (17-24) 4G8 antibody | Biolegend | 800701 | |
| anti-amyloid beta (N terminus 82E1) antibody | IBL America | 10323 | |
| anti-amyloid fibril LOC antibody | EMD Millipore | AB2287 | |
| BCA kit | Thermo Fisher Scientific | 23225 | |
| Bioruptor Pico Plus | Diagenode | B01020001 | |
| Calcium Chloride | Sigma-Aldrich | C1016 | |
| Collagenase | Sigma-Aldrich | C0130 | |
| Complete Protease Inhibitor Cocktail | Sigma-Aldrich | 11697498001 | |
| Dnase I | Thermo Fisher Scientific | EN0521 | |
| EDTA | Sigma-Aldrich | EDS | |
| Guanidine hydrochloride | Sigma-Aldrich | G4505 | |
| HyperSep C18 Cartridges | Thermo Fisher Scientific | 60108-302 | |
| Integrated Proteomics Pipeline – IP2 | http://www.integratedproteomics.com/ | ||
| Iodoacetamide (IAA) | Sigma-Aldrich | I1149 | |
| K54 Tissue Homogenizing System Motor | Cole Parmer | Glas-Col 099C | |
| MaxQuant | https://www.maxquant.org/ | ||
| Micro BCA kit | Thermo Fisher Scientific | 23235 | |
| Nanoviper 75 μm x 50 cm | Thermo Scientific | 164942 | |
| Optima L-90K Ultracentrifuge | Beckman Coulter | BR-8101P-E | |
| Orbitrap Fusion TribridMass Spectrometer | Thermo Scientific | IQLAAEGAAPFADBMBCX | |
| Pierce C18 Spin Columns | Thermo Fisher Scientific | 89870 | |
| Precellys 24 tissue homogenizer | Bertin Instruments | P000062-PEVO0-A | |
| ProteaseMAX(TM) Surfactant Trypsin Enhancer | Promega | V2072 | |
| RawConverter | http://www.fields.scripps.edu/rawconv/ | ||
| Sodium azide | VWR | 97064-646 | |
| Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | 74255 | |
| Sorvall Legend Micro 21R Microcentrifuge | Thermo Fisher Scientific | 75002446 | |
| Speed Vaccum Concentrator | Labconco | 7315021 | |
| Tris-2-carboxyethylphosphine (TCEP) | Sigma-Aldrich | C4706-2G | |
| Tris-HCl | Thermo Fisher Scientific | 15568025 | |
| Trypsin Gold-Mass spec grade | Promega | V5280 | |
| UltiMate 3000 RSLCnano System | Thermo Scientific | ULTIM3000RSLCNANO |
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Citazione di questo articolo
Upadhyay, A., Vassar, R. J., Savas, J. N. Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain. J. Vis. Exp. (182), e63816, doi:10.3791/63816 (2022).