代谢标记和膜分馏的比较蛋白质组学分析<em>拟南芥</em>悬浮细胞培养
Summary
在这里，我们描述了植物细胞质膜的基础上，未标记的混合物分馏成耐洗涤剂和清洁剂的可溶性和膜在体内完全15 N标记的拟南芥细胞培养一个强大的方法。该过程被施加于比较蛋白质组学研究，了解信令进程。
Abstract
质膜微区是基于所述脂质和甾醇环境中的物理性质的功能，并有在信令进程特定角色。从植物细胞中提取甾醇富集的膜微区用于蛋白质组分析是一项艰巨的任务，主要是由于多个制备步骤和来源自其它细胞区室的污染。质膜构成大约只有5-20％的在植物细胞中的所有膜的，因而高度纯化的质膜组分的分离是具有挑战性的。经常使用的方法涉及水性两相分配中的聚乙二醇和葡聚糖，这将产生质膜囊泡为95％1的纯度。质膜内固醇丰富的膜微区是不溶性后用冷的非离子型洗涤剂在碱性pH下处理。此耐洗涤剂膜组分可以从散装质膜中作为分离通过超速离心ucrose梯度2。其后，蛋白质可以提取从由甲醇/氯仿沉淀的蔗糖梯度密度低频段。提取的蛋白质将被胰蛋白酶消化，脱盐，最后用LC-MS/MS分析。我们的提取协议甾醇富集微区被优化用于从拟南芥细胞培养清洁洗涤剂性膜级分的制备方法。
我们利用拟南芥悬浮细胞培养的全代谢标记为K 15 NO 3作为唯一的氮源为后续生物处理的利息3定量比较蛋白质组学研究。通过将标记和未标记的细胞培养物进行联合蛋白质提取的相等比例对最终定量结果的制备步骤的影响被保持在最低限度。还材料的提取过程中的损失将影响控制和以同样的方式处理的样品，一个ð因此光线和升沉肽的比率将保持不变。在所提出的方法无论是标记的或未标记的细胞培养物经历了一个生物处理，而另一作为控制4。
Introduction
1972年，乔纳森歌手和加思尼科尔森提出的流体镶嵌模型细胞膜的结构模型，替换被普遍接受的20世纪60年代初的蛋白质 – 脂质 – 蛋白质三明治模型。歌手和尼科尔森推测，生物膜可以被认为是一个两维的液体，所有的脂质和蛋白质分子的自由和方便5扩散。自那时起，该膜组合物的质膜和知识的结构模型变得更加复杂。特别是，在质膜内，结构，例如蛋白质复合物和脂质/固醇基础结构紊乱的微区可以观察到。在仿真模型膜6,7，固醇和鞘脂可以横向与其他脂类物质分离，形成区域与改变的物理特性。细胞膜内偏析这主要是由甾醇及高度SA之间的自相关联的属性phopsho和鞘脂8的turated烃链。特别是，刚性甾醇环青睐与硬，直饱和脂质种类以及这些相互作用迫使邻近的烃链为更长时间的构象，增加膜的厚度和硬度的相互作用。
之一的甾醇富集的膜微区中通常观察到的特征是其不溶性，在治疗与非离子洗涤剂如曲通X-100或的Brij 35。这些组分被认为是相同的膜微区，并根据其生化制备方法2，被称为耐洗涤剂膜（DRM）。非离子型洗涤剂的DRM提取过程中使用接收到的一些批评作为生化DRM制剂可以不直接对应于活细胞9中的任何特定的膜隔室。特别是，所述洗涤剂蛋白质比率似乎在这类制剂是至关重要的，一s个不同的清洁剂，以及不同量的洗涤剂可以产生不同的耐洗涤剂的膜部分10的组合物。然而，有证据表明，特定蛋白的物种特异性与这些细胞的甾醇富集膜域相关联，并且这些蛋白质以及富含洗涤剂耐膜部分11的生化制剂。被发现在植物的DRM级分，并针对其存在的DRM中是固醇依赖性蛋白质的核心，是特别GPI锚定的蛋白质，如fasciclin状阿拉伯半乳聚糖蛋白（FLAS）和SKU蛋白家族的成员。也有一些信号蛋白，如受体样激酶或磷脂酶被发现11。这些结果与对哺乳动物的膜微区12,13许多蛋白质组研究是一致的。此外，在植物有越来越多的证据表明膜微的应激反应14方面的作用–16。
这里所描述的协议提供了一个可靠的方法对质膜微区分离，特别是使用蛋白质洗涤剂浓度，使我们能够描绘固醇丰富的膜隔4,11,14应激诱导的改变。
Protocol
程序在提取协议中常用的试剂和缓冲液： 喷气推进实验室的培养基拟南芥悬浮细胞培养 3μM的H 3 BO 3 3微米硫酸锰4×H 2 O的 1.1微米的ZnSO 4×7 H 2 O 0.15微米KJ 0.03微米的Na 2的MoO 4×2 H 2 O的 3纳米氯化钴2×6 H 2 O 3纳米硫酸铜4×5 H <sub…
Representative Results
使用代谢标记的拟南芥细胞培养物中所提出的协议，可以从植物组织中（步骤2， 图2和图4）分离血浆膜，血浆膜中富集耐洗涤剂的膜部分（步骤3， 图3和图5）。其后，该协议允许提取从这些耐洗涤剂的膜部分（步骤4）和消化的蛋白质进行比较蛋白质组学分析（步骤5）的蛋白质。最后，磷酸（第8步）的可选的富集是研究细胞信号传导过程特别有趣。最后一步则是?…
Discussion
在本文提出的协议包含了许多步骤，所有的人都非常重要，以获得植物质膜到洗涤剂耐膜和清洁剂的可溶性组分的纯净和有代表性的分馏。因此，作为指示要按照每个步骤是很重要的。
质膜组分与非离子型去污剂（步骤3.2）的治疗有对的膜微区分馏质量最强的影响。以获得可再现的结果不同制剂之间，并以能够从相同的实验比较不同样品，精确地评估蛋白质在细胞膜的浓度?…
Disclosures
The authors have nothing to disclose.
Materials
| REAGENTS | |||
| Chemicals were ordered from Sigma-Aldrich unless noted otherwise | |||
| Ammonia (stock 25 % solution) | WAKO | 010-03166 | |
| TiO2 10 μm | GL-Science | 5020-75010 | |
| Empore Disk C18 | Varian | 12145004 | |
| Polyethylene glycol(PEG 3350) | Sigma | 88276 | |
| Dextran T500 | Roth | 9219.2 | |
| Trypsin | Promega | V5113 | |
| Protease inhibitor cocktail (PIC) | Sigma | P9599 | |
| K15NO3 | Cambridge Isotope Laboratories | NLM-765-PK | |
| EQUIPMENT | |||
| Optima L-80 XP Ultracentrifuge | Beckman | ||
| Plate reader | BioTek | ||
| EASY-nLC II nano-Liquid Chromatograph | Thermo Scientific | ||
| LTQ-Orbitrap mass spectrometer | Thermo Scientific | ||
| Centrifuge 5810R | Eppendorf | ||
| Centrifuge 5417R | Eppendorf | ||
| Thermomixer | Eppendorf | ||
| Speed Vac RVC 2-25 | Christ | ||
| Shaker Unimax 2010 | Heidolph |
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Cite This Article
Szymanski, W. G., Kierszniowska, S., Schulze, W. X. Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures. J. Vis. Exp. (79), e50535, doi:10.3791/50535 (2013).