MALDI sample preparation: the ultra thin layer method.
MALDI sample preparation: the ultra thin layer method.
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DOI：
10.3791/192
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发表时间：
2007-01-01
期刊：
影响因子：
--
通讯作者：
Chait, Brian T
中科院分区：
文献类型：
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作者：
Fenyo, David;Wang, Qingjun;Chait, Brian T
该视频演示了如何制备超薄基质/分析物层，用于通过基质辅助激光解吸电离质谱 (MALDI-MS) 分析肽和蛋白质 (1, 2)。超薄层方法涉及在样品板上生成基质晶体（α-氰基-4-羟基肉桂酸）基质层，作为基质/分析物混合物后续结晶的播种地。与其他样品沉积方法（例如干燥液滴）相比，超薄层方法的优点在于：（i）对盐和清洁剂等杂质具有更大的耐受性，（ii）更好的分辨率，以及（iii）更高的空间均匀性。该方法对于蛋白质的精确质量测定特别有用。该方案最初是为分析膜蛋白而开发和优化的，并用于成功分析包含 2 至 12 个跨膜结构域的离子通道、代谢物转运蛋白和受体 (2)。自最初发表以来，它也被证明对于可溶性蛋白质的分析同样有用。事实上，我们已将其用于大量具有多种特性的蛋白质，包括那些分子量高达 380 kDa 的蛋白质 (3)。目前，它是我们对所有蛋白质进行分子质量分析的首选方法。所描述的程序始终能够产生高质量的光谱，并且灵敏、稳健且易于实施。
This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) (1, 2). The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains (2). Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa (3). It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.