Name: t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis
Uploaded: 2010-07-31T17:00:00
Duration: 16 min 40 s
Description: Watch this Scientific Journal Video about T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis at JoVE.com

作者要感谢他们的严格审查，并为他们贡献的手稿沙龙组的成员。我们Morasha和Bikura方案，以色列科学基金会（批准号：1823至1807年和378/08），约瑟夫科恩密涅瓦生物膜研究中心，翟氏父子家庭研究员新科学家计划，亚伯拉罕的支持表示感谢索尼娅Rochlin基金会，欧胜家庭慈善信托基金的海伦和米尔顿答Kimmelman中心生物分子结构及组装;什洛莫和萨宾Beirzwinsky房地产; Meil​​德波顿Aynsley，和Karen暹粒，英国。

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这里所描述的协议使定义一个未知的三维结构的蛋白质或蛋白质复合物的碰撞截面，其整体造型，包装亚基和拓扑提供信息的目的。为此，一旦碰撞截面值描述有必要将这些值转换结构的细节。这个过程，需要额外的实验努力，以及计算分析，下面简要讨论。 首先，它是建议进行分析与已知结构的蛋白质或蛋白质复合物。这些测量数据可以提供一个有用的质量控制的方法，将使比较Ω理论和测量值的采集参数的精度评估。可以计算出晶体结构的理论截面领域坐标使用MOBCAL 15,16软件，它是一个开放源码的FORTRAN为基础的软件，根据运营商需要允许代码编辑。运行这样的计算，它是需要修改的方案等，每输入结构进行迭代计算的数量增加，并含有大量原子的坐标文件被接受1。 IM - MS的战略定义拓扑安排在多元集会亚基最近已建议4,6。该方法涉及的蛋白质装配的分解途径，以更小的元件监测。这种分解是通过调整控制的解决方案相条件下，这就会引起subcomplexes的“积木”的集会反射分布。完整复杂和拆卸产品的Ω值同时测量计算的蛋白质复合物的拓扑模型，然后使用生成的结构限制。这种方法的基本假设是产生subcomplexes保留自己的母语一样确认，而事实上最近的研究表明，维护和拆装产品的解决方案的结构是没有任何一个解决方案或气体阶段的主要重排发生4,6。 气相蛋白复合物离子的四级结构的分配中的最后一步是计算机生成的模型拟合​​的碰撞截面值。建模方法，以探讨亚基不同的可能的拓扑安排和他们在硅片 Ω值的计算和比较实验。目前只有少数的计算方法是，像spheretype粗粒度的方法，直径接近亚基1,8。整体而言，这一领域仍然是在它的早期，并进一步发展的需要，使这种方法是通用的，适用于广泛的复合物。

t-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis

离子淌度（IM）是一种方法，措施离子穿过一个微弱的电场的影响下加压细胞所需的时间。离子穿过漂移区的速度，这取决于它们的大小：大离子的经验与背景的惰性气体（ 通常 N 2）将更多的碰撞，从而比那些包括一个较小的的离子穿过的IM设备更慢的横截面。在一般情况下，的时间，需要的离子迁移，虽然稠密气体相分离，根据他们的碰撞截面（Ω）。 近日，IM法加上质谱和一个行波（T波）SYNAPT离子迁移质谱仪（IM - MS）被释放。离子迁移质谱相结合，使样品分离和定义一个额外的维度，产生一个三维频谱（质量负责，强度和漂移时间）。此分离技术使光谱重叠，以减少，质量非常相似，或质荷比，但不同的漂移时间，使异构配合物的决议。此外，漂移时间的测量提供了一个重要的结构信息层，Ω是与离子的整体形状和拓扑。定义截面1校准蛋白质产生的校正曲线计算测得的漂移时间值和Ω之间的相关性。 的IM - MS方法的力量在于它能够定义亚基在微摩尔浓度的蛋白质集会的包装和整体造型， 近生理条件1。几个最近2,3个人的蛋白质和非共价键蛋白质复合物 4-9 IM的研究，成功地证明，蛋白质的四级结构是保持在气相，并强调这种方法的潜力，在未知的几何形状的蛋白质集会的研究。在这里，我们提供的IMS - MS分析的蛋白复合物使用的SYNAPT（四极离子迁移的飞行时间）HDMS仪器（沃特公司;目前唯一的商业IM - MS仪器）详细的说明10。我们描述的基本优化步骤，碰撞截面校准，数据处理和解释方法。该协议的最后一步讨论理论Ω值计算的方法。总体而言，该协议并不试图掩盖IM - MS的表征蛋白质装配的每一个方面，而是它的目标是引进在该领域的新的研究方法的实际问题。

Watch this Scientific Journal Video about T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis at JoVE.com

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

离子迁移质谱是一个新兴的技术，分离离子气相，他们的碰撞截面和质量的基础上。该方法提供了对整体的拓扑结构和蛋白质复合物的形状的三维信息。在这里，我们勾勒出一个仪器设置和优化，校准漂移的时间，和数据解释的基本程序。

T波离子迁移质谱蛋白复合物分析的基本实验程序

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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