Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products
Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products
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DOI：
10.1002/rcm.4800
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发表时间：
2010-12-01
影响因子：
2
通讯作者：
Fenaille, Francois
中科院分区：
文献类型：
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作者：
Manduzio, Helene;Ezan, Eric;Fenaille, Francois
我们研究了聚合酶链式反应(PCR)和电喷雾电离质谱仪(ESI-MS)离线联用的潜力和稳定性，为进一步应用于单核苷酸多态(SNPs)的筛查奠定了基础。这是基于最近报道的数据，该数据表明，阴离子交换固相萃取是有效脱盐PCR产物的最有效技术，回收率接近70%。结果表明，这种纯化方法有效地去除了几乎所有通常添加到PCR缓冲液中的化学物质。在LTQ-Orbitrap仪器上进行的114-BP聚合酶链式反应产物的ESI-MS分析表明，使用外部校准可以常规获得NM范围内的检测下限以及9.15+/-7.11ppm的平均质量测量不确定度。PCR/ESI-MS平台只能检测到目标寡核苷酸的几个拷贝。然而，研究表明，如果两个扩增产物以高于10：1的比例存在于混合物中，则可能无法重复检测到丰度较低的一个。对SNPs的应用表明，分辨率为30 000(m/z 400)的LTQ-Orbitrap很容易识别类似于35 kDa聚合酶链式反应产物的二元混合物中的单个(A G)开关，即16Da的差异。互补实验也表明，内切酶和ESI-MS的结合可以用来确定碱基组成和序列，从而筛选特定序列中的未知多态。例如，在114bp的聚合酶链式反应产物中成功地鉴定了单个(T A)开关(9Da质量差)。版权所有(C)2010 John Wiley&Sons，Ltd.
We have investigated the potential and robustness of the off-line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI-MS), for further applications in the screening of single-nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion-exchange solid-phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of similar to 70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI-MS analysis of a model 114-bp PCR product performed on the LTQ-Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 +/- 7.11ppm can be routinely obtained using an external calibration. The PCR/ESI-MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ-Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A G) switch, i.e. a 16 Da difference, in binary mixtures of similar to 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI-MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T A) switch (9 Da mass difference) was successfully identified in a 114-bp PCR product. Copyright (c) 2010 John Wiley & Sons, Ltd.