Wu H, Chan C, Aboleneen H
Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA.
Anal Biochem. 1998 Oct 15;263(2):129-38. doi: 10.1006/abio.1998.2812.
A method using a combination of enzymatic digestion and ionspray mass spectrometry (MS) was developed for sequencing oligodeoxynucleotides (ODNs) containing more than 20 bases. Phosphodiesterase (PDE) digestion of ODNs produced truncated ODNs whose molecular weights (MWs) were determined by ionspray MS. It was demonstrated that reconstruction of MW spectra over a large MW range produced easy-to-read sequence ladders similar to those obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Sample and enzyme cleanup, digestion control, and MW reconstruction were found to be crucial factors. For regular ODNs, both 5'- and 3'-PDE digestions are needed for complete sequencing. Late in the time course of PDE digestions, 5'-nucleoside monophosphates were found to produce artifactual peaks in the reconstructed MW spectra, and a table correlating base compositions and MS ions was compiled to handle such situations. For labeled ODNs, it is necessary to use collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) for complete sequence determination. Sequencing of regular 22-mer and labeled 18-mer ODNs was demonstrated in this work.
开发了一种结合酶消化和离子喷雾质谱（MS）的方法，用于对含20多个碱基的寡脱氧核苷酸（ODN）进行测序。ODN经磷酸二酯酶（PDE）消化产生截短的ODN，其分子量（MW）通过离子喷雾MS测定。结果表明，在较大MW范围内重建MW谱会产生易于读取的序列阶梯，类似于使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）获得的序列阶梯。发现样品和酶的净化、消化控制以及MW重建是关键因素。对于常规ODN，完整测序需要进行5'-和3'-PDE消化。在PDE消化过程后期，发现5'-单磷酸核苷会在重建的MW谱中产生假峰，并编制了一个将碱基组成与MS离子相关联的表格来处理这种情况。对于标记的ODN，必须使用碰撞诱导解离串联质谱（CID-MS/MS）来进行完整的序列测定。这项工作展示了对常规22聚体和标记的18聚体ODN的测序。
