Ragas J A, Simmons T A, Limbach P A
Department of Chemistry, Louisiana State University, Baton Rouge 70803, USA.
Analyst. 2000 Apr;125(4):575-81. doi: 10.1039/a909709k.
Metal adducts (e.g., Na+ and K+) significantly hinder the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although a number of sample purification methods exist, to date no comparative study exists to determine the most efficient method for purifying oligonucleotides. The objective of this work was to perform such a study. Several different oligonucleotide samples were synthesized. Aliquots of these samples were then purposely contaminated with sodium acetate to generate representative contaminated (salted) oligonucleotide samples. A number of popular oligonucleotide purification techniques were then tested as to their effectiveness at removing Na+ from the salted samples. The effectiveness of Na+ removal was qualitatively assessed by comparing the MALDI mass spectra of the original sample, the salted sample, and the salted sample after purification. Micropipet tips packed with C18 reversed-phase packing material (e.g., Zip Tips) appear to be the most effective means of purifying the oligonucleotides investigated. Minidialysis was found to be an effective alternative for purifying higher molecular weight oligonucleotides (> 10,000 u).
金属加合物（如Na⁺和K⁺）会显著阻碍基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）对寡核苷酸的分析。尽管存在多种样品纯化方法，但迄今为止尚无比较研究来确定纯化寡核苷酸的最有效方法。这项工作的目的就是进行这样一项研究。合成了几种不同的寡核苷酸样品。然后将这些样品的等分试样特意用醋酸钠污染，以生成具有代表性的受污染（含盐）寡核苷酸样品。接着测试了多种常用的寡核苷酸纯化技术在从含盐样品中去除Na⁺方面的有效性。通过比较原始样品、含盐样品以及纯化后含盐样品的MALDI质谱，对Na⁺去除的有效性进行了定性评估。填充有C18反相填充材料的微量移液器吸头（如Zip吸头）似乎是纯化所研究寡核苷酸的最有效方法。发现微量透析是纯化较高分子量寡核苷酸（>10,000 u）的有效替代方法。
