亲水作用色谱法
化学
色谱法
质谱法
寡核苷酸
试剂
高效液相色谱法
生物化学
有机化学
DNA
作者
Bingchuan Wei,Jenny Wang,Lulu Dai,Bifan Chen,Kelly Zhang
标识
DOI:10.1016/j.chroma.2023.464414
摘要
In this study, we aimed to develop a hydrophilic interaction liquid chromatography (HILIC) method for the analysis of single guide ribonucleic acid (sgRNA), a critical reagent used in CRISPR genome editing. Our results showed that effective profiling of sgRNA can be achieved by suppressing the surface charge of the stationary phase in HILIC. We identified hydrogen bonding as the primary retention mechanism with potential weak partitioning in HILIC separation of large oligonucleotides like 100-mer sgRNA. Moreover, we demonstrated that direct coupling of HILIC with mass spectrometry (MS) allows the intact mass analysis of sgRNA and its impurities with minimal adduct present. Finally, we characterized the post peak shown in the low temperature HILIC and identified it as sgRNA aggregates. Our findings provide valuable insight into the characterization of sgRNA and highlight the potential of HILIC-MS as a powerful analytical tool for relatively large oligonucleotide analysis.
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