Name: enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry
Uploaded: 2018-05-21T12:30:00
Description: Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

在梅瑞狄斯实验室的研究由埃伯利科学学院 (宾夕法尼亚州立大学) 提供的启动资金支持。我们感谢 Laremore 博士的专家技术咨询和获得设备在宾夕法尼亚州立大学的蛋白质组学和质谱的核心设施, 学院公园, PA, 在那里进行质谱分析。

1. 细胞生长和裂解
<ol><li>在15毫升胰蛋白酶大豆肉汤中生长细菌或类似的富媒体到晚期指数相 (OD600 1.0-1.5)。用离心法收割细胞, 用三缓冲盐水/EDTA (TBSE) 冲洗一次, 并继续使用协议或冷冻直至用途。 注: TBSE:20 毫米三盐酸盐 (HCl), pH 值 8.0, 130 毫米氯化钠 (氯化钠), 和5毫米乙二胺乙酸 (EDTA))。脂蛋白表达和修饰可能受生长条件 (例如酸度、盐度、生长介质) 和生长阶段15</s...

<ol>
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本协议描述了两个不同阶段的脂蛋白表征: 富集 TX-114 相分配和结构确定的 MALDI MS。在 TX-114 萃取过程中, 额外的离心去除了在这个过程中沉淀的蛋白质, 其次是丙酮沉淀产生高浓度脂蛋白。通过将每个准备的规模限制为15毫升的细胞, 可以很容易地并行处理多个样本, 如果需要, 则在协议结束时将它们汇集在一起。
对于 MS 的结构分析, 将脂蛋白转移到硝化纤维素, 有利于胰蛋白酶...

细菌脂蛋白的特点是保守的 n-末端脂修饰半胱氨酸, 锚定球形蛋白领域的细胞膜表面。它们是普遍分布在细菌, 构成2到5% 的所有细胞基因在一个典型的基因组1。脂蛋白在多种细胞过程中起关键作用, 包括养分吸收、信号传导、蛋白质复合体的组装以及维持细胞包络结构完整性2。在致病性细菌中, 脂蛋白作为致病因子3,4。在感染过程中, 通过类似于收费受体 (TLR) 2 对 n-端脂肽的识别, 激发了先天免疫应答, 以清除入侵的病原体。根据 n-端酰化态, 脂蛋白通常由交替 TLR2 heterodimeric 配合物识别。TLR2-TLR1 承认 n-酰脂肽, 而 TLR2-TLR6 束缚自由脂肽α氨基终点。一旦绑定, 信号通路汇聚以诱导分泌促炎细胞因子3,4。
以前认为, 革兰氏阳性菌脂蛋白 diacylated, 革兰阴性菌 triacylated, 不同于不存在或存在酰胺相关脂肪酸的保守 n-端半胱氨酸残留。这种假设支持的是缺乏序列 orthologs 的革兰阳性基因组 Lnt, 革兰阴性 n-酰基转移酶, 形成 triacylated 脂蛋白5。然而, 最近的研究揭示了 Firmicutes 中缺乏lnt的脂蛋白 triacylation, 以及三种新的 n-端脂蛋白结构, 称为 peptidyl、溶血和n-乙酰形式67 ,8。这些发现引发了对可能尚未发现的脂蛋白形态的质疑, 还有关于这些新脂蛋白是如何制作的基本问题, 以及各种形式所传授的生理目的或优势。此外, 他们清楚地表明, 目前基因组无法预测脂蛋白结构。事实上, 我们最近发现了一种新的类脂蛋白 n-酰转移酶, 称为点燃, 从粪肠球菌和蜡样芽孢杆菌, 使溶血脂蛋白 9.这表明需要实验性地验证脂蛋白结构, 这可能是挑战, 由于其极其疏水性的性质和有限的方法, 以表征其分子结构。
为了促进对脂蛋白诱导宿主免疫应答以及 n 末端结构测定的研究, 我们已经修改了几种先前描述的协议, 以纯化细菌脂蛋白并制备 n-端胰蛋白酶。脂肽用于分析的 MALDI MS 6, 10, 11, 12.脂蛋白是丰富使用建立的海卫 X-114 (从今以后称为表面活性剂或 TX-114) 相分配方法, 优化去除污染的非脂蛋白和增加脂蛋白产量。这些脂蛋白适用于直接使用 TLR 化验或进一步纯化的 SDS 页。对于 MALDI MS, 脂蛋白转移到硝化棉膜提供了一个脚手架, 有效的原位胰蛋白酶消化, 洗涤, 并随后从膜表面洗脱, 导致高度纯化的 n-端脂肽。硝化棉已被证明可以促进样品处理和改善序列覆盖的高度疏水性肽从整体膜蛋白13,14, 以及脂蛋白9,10.该方法具有以极性为基础的分馏肽的额外优势, 在单实验中, 可以分析中间洗涤液与 n-末端结构测定同时进行高置信蛋白鉴定。.该协议独特的特点是有意的钠加成层, 以促进在 ms/毫秒内的父离子分裂为 dehydroalanyl 离子, 协助结构分配的N-酰化态。N 终点是与 TLR 识别脂蛋白有关的最大变量和关键特征。总之, 本议定书允许对脂蛋白进行深入和可重复性的研究, MALDI 的纯化和结构测定的各个阶段根据实验的总体目标容易适应。

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 mm Zirconia/silica beads</td>
			<td>BioSpec Products</td>
			<td>110791012</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMD</td>
			<td>AX0116-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0142-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fluka Analytical</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTrace NT Nitrocellulose</td>
			<td>PALL Life Sciences</td>
			<td>66485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) digest standard</td>
			<td>Protea</td>
			<td>PS-204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade water</td>
			<td>EMD</td>
			<td>WX0008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Fisher Scientific</td>
			<td>BP535-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethyl sulfonyl fluoride (PMSF)</td>
			<td>Amresco</td>
			<td>0754</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce trypsin protease</td>
			<td>Thermo Scientific</td>
			<td>90057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Acros Organics</td>
			<td>161470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5mL</td>
			<td>Eppendorf</td>
			<td>022431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>792519</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macron Fine Chemicals</td>
			<td>7581-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-hydrochloride (HCl)</td>
			<td>Fisher Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-114</td>
			<td>Sigma-Aldrich</td>
			<td>93422</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>BD</td>
			<td>211822</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid (MS Grade)</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagNALyser</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOF target</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraflextreme MALDI-TOF-TOF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Equipped with a 355 nm frequency-tripled Nd:YAG smartbeam-II laser</td>
		</tr>
	</tbody>
</table>

enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry

脂蛋白是哺乳动物先天免疫应答的细菌细胞包络和有效活化剂的重要成分。尽管它们对细胞生理学和免疫学都有重要意义, 但对于新的脂蛋白形态、合成方法以及各种形式对宿主免疫的影响, 仍有许多有待发现。为了对脂蛋白进行深入研究, 本协议描述了一种细菌脂蛋白的富集方法和 n-端胰蛋白酶脂肽的制备, 其结构由基质辅助激光解吸电离-飞行时间来确定。质谱 (MALDI)。在建立的海卫 X-114 相分治法中扩展了从细菌细胞膜中提取和富集的方法, 该协议包括去除非脂蛋白污染物的附加步骤, 增加脂蛋白产量和纯度。由于脂蛋白是常用的收费样受体 (TLR) 检测, 这是至关重要的第一个特点的 n-终端结构的 MALDI 的 MS. 本文提出了一种分离富集在 n-端的浓疏水性多肽的方法。脂肽适合直接分析的 MALDI 女士/ms. 经月桂酸钠分离的脂质体-丙烯酰胺凝胶电泳 (SDS 页) 被转移到硝化纤维膜上, 消化原位 胰蛋白酶, 依次洗涤去除极性胰蛋白酶肽, 最后洗脱与氯仿-甲醇。当与洗涤液中极性 trypsinized 肽的 MS 结合时, 这种方法提供了在单个实验中识别脂蛋白并表征其 n-终点的能力。故意的钠加成物也可以作为一种工具, 以促进更结构化的信息碎片谱。最终, 脂蛋白的丰富和对其 n-端结构的测定将允许对这种无处不在的细菌蛋白进行更广泛的研究。

协议的示意图在图 1中提供。图 2中显示了从粪肠球菌ATCC 19433 中提取的富含脂蛋白的分数 TX-114。为比较, 还显示了沉淀蛋白分数的带状模式。从这个分数的蛋白质证实了 MALDI-MS 是高度丰富的污染蛋白质以外的脂蛋白 (表 1)。在图 3中的质谱显示了 PnrA 的胰蛋白酶肽离子剖面, 该蛋白是在?...

Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

用非离子表面活性剂相分配法对细菌脂蛋白的富集进行了描述, 直接用于 TLR 化验或其他应用。进一步详细的步骤, 以准备 n-终端胰蛋白酶脂肽的结构表征的质谱。

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

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