Dietrich Susanne, Dollinger Anja, Wieser Andreas, Haisch Christoph
Chair of Analytical Chemistry, TUM School of Natural Sciences, Technical University of Munich, Munich, Germany.
Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Immunology, Infection and Pandemic Research, Frankfurt, Germany.
Rapid Commun Mass Spectrom. 2025 Aug 15;39(15):e10061. doi: 10.1002/rcm.10061.
The reproducibility of the analysis of oligonucleotides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) remains a significant challenge. This is mainly attributed to factors such as the choice and application of the matrix, as well as the inhomogeneity of the sample spots. Although previous studies have explored various parameters, such as ionic and sugar-based matrices, and different spotting methods, none have comprehensively integrated these factors into a unified approach.
Various matrices for the analysis of oligonucleotides and the effects of diverse variables, including different matrices, solvent compositions, additives, and application techniques, on the analytical performance of these matrices were investigated. Mass spectrometry analysis was conducted by a MALDI-TOF MS in linear negative mode.
Our research systematically evaluates the combined effects of diverse variables to enhance the analytical performance of MALDI-TOF MS in oligonucleotide analysis. We focused on the standard deviation of mass-to-charge ratios and the signal-to-noise (S/N) ratios. Out of 48 samples, only 19 met the S/N criteria, which is that the signals must be detectable over the whole mass range of interest (4-10 kDa).
The ionic matrix 6-aza-2-thiothymine (ATT) with 1-methylimidazole resulted consistently in a reduced standard deviation and achieved high mass precisions. Additionally, we observed that the S/N ratios and mass precision of 3-hydroxypicolinic acid (3-HPA) varied significantly depending on the solvent composition and the presence of additives.
使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析寡核苷酸的重现性仍然是一项重大挑战。这主要归因于诸如基质的选择和应用以及样品点的不均匀性等因素。尽管先前的研究已经探索了各种参数，如离子型和糖基型基质以及不同的点样方法，但没有一项研究将这些因素全面整合到一种统一的方法中。
研究了用于寡核苷酸分析的各种基质以及包括不同基质、溶剂组成、添加剂和应用技术在内的各种变量对这些基质分析性能的影响。通过线性负模式的MALDI-TOF MS进行质谱分析。
我们的研究系统地评估了各种变量的综合影响，以提高MALDI-TOF MS在寡核苷酸分析中的分析性能。我们关注质荷比的标准偏差和信噪比（S/N）。在48个样品中，只有19个符合S/N标准，即信号必须在整个感兴趣的质量范围内（4-10 kDa）可检测到。
离子型基质6-氮杂-2-硫代胸腺嘧啶（ATT）与1-甲基咪唑一起使用时，始终能降低标准偏差并实现高质量精度。此外，我们观察到3-羟基吡啶甲酸（3-HPA）的信噪比和质量精度会因溶剂组成和添加剂的存在而有显著差异。
