Biomedical Microscale Analytics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, Leiden, The Netherlands.
Netherlands Metabolomics Centre, Leiden, The Netherlands.
Electrophoresis. 2020 Mar;41(5-6):360-369. doi: 10.1002/elps.201900417. Epub 2020 Jan 13.
Negative ion mode nano-ESI-MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano-ESI needle emitter lifetimes. In this work, we introduce a sheathless CE-MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16 mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono-, di-, and tri-phosphates, could be efficiently analyzed with plate numbers per meter above 220 000 and with LODs in the range from 0.06 to 1.3 nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50 000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide-adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material.
负离子模式纳喷雾电喷雾质谱法（nano-ESI-MS）常用于分析酸性化合物，包括核苷酸。然而，在高水相分离条件下，发射尖端经常会发生电晕放电，这可能导致离子丰度低，纳喷雾针发射极寿命缩短。在这项工作中，我们介绍了一种无鞘毛细管电泳-质谱联用（CE-MS）方法，用于采用正离子模式的高效、灵敏分析核苷酸，从而完全避免电晕放电。使用背景电解质为 16 mM 乙酸铵（pH 9.7），可以有效地分析由单、二和三磷酸盐组成的 12 种核苷酸混合物，理论塔板数超过 220000，检测限（LOD）范围为 0.06 至 1.3 nM，相当于 0.4 至 8.6 阿托摩尔，仅需进样体积约 6.5 nL。该方法的实用性通过使用 HepG2 细胞作为模型系统，对低数量哺乳动物细胞中的核苷酸进行分析得到了验证。仅从 50000 至 500 个 HepG2 细胞的提取物中，就可以有效地分析内源性核苷酸。此外，除了核苷酸之外，在这些条件下还可以分析一些烟酰胺腺嘌呤二核苷酸和氨基酸，从而清楚地说明了该方法用于低量生物材料代谢分析的实用性。
