A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry
A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry
复制标题
DOI：
10.1016/s0003-2697(03)00024-1
复制
发表时间：
2003-05-01
影响因子：
2.9
通讯作者：
Hofstadler, SA
中科院分区：
文献类型：
--
作者：
Jiang, Y;Hofstadler, SA
在这项工作中，我们提出了一种快速和全自动化的方法来纯化和脱盐PCR产物，然后通过电喷雾电离质谱分析。该方案采用了一个商业移液器吸头填充有阴离子交换树脂，并包括四个主要步骤：吸头预处理，样品加载，冲洗和样品洗脱。这种基于尖端的纯化/纯化方案与先前公开的方法相比具有两个明显的优点。首先，该方案可以使用标准p10手动移液器手动进行（每次1 - 12个样品），或者采用标准实验室机器人技术以全自动微量滴定板形式进行（每次96个样品）。此外，从粗PCR产物到"电喷雾"分析物溶液的整个方案仅需要10穆尔粗产物，并且需要不到20 min。使用毛细管凝胶电泳，我们证明了类似于80%的总体回收率，并且证明了高性能电喷雾电离傅立叶变换离子回旋共振质谱的精致的分离效率。使用内部质量标准，我们证明了亚ppm质量测量误差，这为120-mer PCR产物提供了明确的基础组成。(C)2003 Elsevier Science（美国）。All rights reserved.
In this work we present a rapid and fully automated method to purify and desalt PCR products prior to analysis by electrospray ionization mass spectrometry. The protocol employs a commercial pipette tip packed with an anion-exchange resin and comprises four primary steps: tip pretreatment, sample loading, rinsing, and sample elution. This tip-based purification/desalting protocol has two distinct advantages over previously published methods. First, the protocol can be performed either manually (1-12 samples at a time), using a standard p10 manual pipette, or in a fully automated microtiter plate format (96 samples at a time) employing standard laboratory robotics. Additionally, the entire protocol from crude PCR product to an "electrosprayable" analyte solution requires only 10 mul of crude product and takes less than 20 min. Using capillary gel electrophoresis, we demonstrate an overall recovery efficiency of similar to80% and demonstrate the exquisite desalting efficiency with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Using an internal mass standard we demonstrate sub-ppm mass measurement error which provides an unambiguous base composition for a 120-mer PCR product. (C) 2003 Elsevier Science (USA). All rights reserved.