Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction
Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction
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DOI：
10.1038/nmeth1145
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发表时间：
2008-01-01
期刊：
影响因子：
48
通讯作者：
Caprioli, Richard M.
中科院分区：
文献类型：
--
作者：
Andersson, Malin;Groseclose, M. Reid;Caprioli, Richard M.
由于大量的基因组和蛋白质组数据集是从各种组织的匀浆中产生的，因此对其编码产物的空间定位信息的需求变得更加迫切。基质辅助激光解吸电离（MALDI）成像质谱（IMS）为研究人员提供了明确研究具有分子特异性的肽和蛋白质并确定其二维和三维分布的方法（1，2）。在过去几年中，对国际监测系统的若干参数进行了优化，包括样品制备、基质应用和仪器采集参数（3、4）（方框1）。这些发展使得质量准确度和峰强度具有高度的重现性（在线补充图1）。最近，我们已经优化了我们的协议，能够增加分子种类的数量分析收集两组部分，覆盖一组部分与芥子酸的蛋白质和相邻部分与2，5-二羟基苯甲酸（DHB）矩阵的最佳检测低质量的物种，包括肽的最佳检测。在每个数据集中可以观察到大约1，000个峰（图1）。此外，在相等的距离，200 μ m，而不是400-500 μ m以前使用的部分收集，从而使虚拟z-堆栈和三维（3D）体积渲染的使用，以调查差异定位模式在更小的大脑结构，如黑质和脚间核。在这里，我们提出了我们的优化的一步一步的程序的基础上，以前的工作在我们的实验室（2），描述了如何使三维体积重建的MALDI IMS数据，适用于大鼠大脑。
As large genomic and proteomic datasets are generated from homogenates of various tissues, the need for information on the spatial localization of their encoded products has become more pressing. Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) offers investigators the means with which to unambiguously study peptides and proteins with molecular specificity, and to determine their distribution in two and three dimensions(1,2). In the past few years, several parameters have been optimized for IMS, including sample preparation, matrix application and instrumental acquisition parameters(3,4) (Box 1). These developments have resulted in a high degree of reproducibility in mass accuracy and peak intensities (Supplementary Fig. 1 online). Recently, we have optimized our protocol to be able to increase the number of molecular species analyzed by collecting two sets of sections, covering one set of sections with sinapinic acid for optimal detection of proteins and adjacent sections with 2,5-dihydroxybenzoic acid (DHB) matrix for the optimal detection of low-mass species, including peptides. Approximately 1,000 peaks can be observed in each dataset (Fig. 1). Furthermore, the sections are collected at an equal distance, 200 mu m instead of 400-500 mu m used previously, thus enabling the use of virtual z-stacks and three-dimensional (3D) volume renderings to investigate differential localization patterns in much smaller brain structures such as the substantia nigra and the interpeduncular nucleus. Here we present our optimized step-by-step procedure based on previous work in our laboratory(2), describing how to make 3D volume reconstructions of MALDI IMS data, as applied to the rat brain.