Wu H, Aboleneen H
Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA.
Anal Biochem. 2001 Mar;290(2):347-52. doi: 10.1006/abio.2001.4993.
The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.
核酸外切酶消化与质谱联用已被广泛用于寡核苷酸测序。在核酸外切酶消化过程中，核苷酸浓度的快速增加会在电喷雾电离质谱中产生强烈的核苷酸簇离子信号，尤其是对于碱基超过25个的寡核苷酸。由于核苷酸簇离子产生的伪峰，这导致后期消化产物的重构分子量谱中的信噪比很差。在此，我们报告一种消除簇离子影响的方法。在该方法中，将碱性磷酸酶与蛇毒磷酸二酯酶加入到寡核苷酸溶液中，将干扰性核苷酸转化为非干扰性核苷，并获得消化结束时二聚体寡核苷酸的碰撞诱导解离谱，以确定寡核苷酸5'-末端最后两个碱基的序列。通过这种方法，对于相对较大的寡核苷酸，重构分子量谱的信噪比得到了极大提高，并且测序仅需一次消化。
