利用MALDI-TOF MS分析寡核苷酸钠盐组分分布  

Analysis of the Distribution of Sodium Oligonucleotides by MALDI-TOF MS

作　　者：黄骏齐 陈尚坤 刘巧霞 HUANG Jun-qi;CHEN Shang-kun;LIU Qiao-xia(Shanghai Medical College of Fudan University,Shanghai 200032,China;Shimadzu Enterprise Management(China)Co.Ltd.,Shanghai 200233,China)

机构地区：[1]复旦大学上海医学院,上海200032 [2]岛津企业管理(中国)有限公司,上海200233

出　　处：《分析测试学报》2025年第4期620-627,共8页Journal of Instrumental Analysis

摘　　要：建立了基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)分析寡核苷酸钠盐分布的方法。单链寡核苷酸引物样品(长度为20 bp,理论分子量6071.02 Da)经纯水溶解后,分别采用高效液相色谱-质谱(HPLC-MS)与MALDI-TOF MS进行检验;样液经乙酸钠加钠、醇沉、复溶后再次以HPLC-MS与MALDITOF MS进行检验,检验模式均为正离子。加钠前后,HPLC-MS在解卷积后均仅显示未加盐的单一组分(对应分子量6069.9 Da与6069.8 Da);MALDI-TOF MS对加盐前样品显示单一组分(对应分子量6071.09 Da),而对加盐后样品显示了不同加钠数分子的组分分布:各组分从未加钠分子开始(对应分子量6071 Da左右),含量呈现先上升后下降的趋势,加钠个数范围从0~4到0~19不等,且分布随加盐条件的不同而变化。该结果显示MALDI-TOF MS具有检测寡核苷酸钠盐组分分布的能力,同时其具有检测一系列不同加盐程度寡核苷酸分子的广泛适应性与灵敏性。以反义寡核苷酸药物福米韦森钠为样本,采用MALDI-TOF MS分别检测了福米韦森钠纯品以及含聚乙二醇和吐温等辅料的福米韦森钠动物注射液模型。结果显示,福米韦森钠的主体加钠形态为加0个钠到加3个钠,整体含量呈现递减趋势,在加入基质前后未发生改变;同时福米韦森钠组分的误差也处于±0.5 Da范围内,精确性未受基质影响。另外,基质中的其他若干组分(包括聚乙二醇300与吐温-80)也独立显示在质谱图中,对福米韦森钠组分没有干扰。该法简单快速、灵敏度高,适用于一系列寡核苷酸钠盐分布的定性鉴定,且检验实际药物时不受药物基质影响,对反义寡核苷酸(ASO)药物的研发与检验有重要意义。A method has been developed for the analysis of sodium distribution of oligonucleotides using matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).The single strand oligonucleotide primers(with a length of 20 bp,whose theoretical molecular weight is 6071.02 Da)were dissolved in pure water and tested by traditional high performance liquid chromatogen-mass spectrometry(HPLC-MS)and MALDI-TOF MS respectively.The sample solution was tested by HPLC-MS and MALDI-TOF MS respectively after sodium acetate and sodium,alcohol precipitation and resolution,and the test mode was positive ion.Before and after sodium addition,HPLC-MS showed only the unsalted single component(corresponding molecular weight 6069.9 Da and 6069.8 Da)after deconvolution.MALDI-TOF MS showed a single component(corresponding molecular weight 6071.09 Da)for the samples before salt addition,while the component distribution of different sodium number molecules was shown for the samples after salt addition:Each component started with no sodium molecules(corresponding molecular weight of about 6071 Da),and its content showed a trend of first increasing and then decreasing,the number of sodium added ranges from 0-4 to 0-19,and the distribution changed with different salt adding conditions.The results showed that MALDI-TOF MS had the ability to detect the distribution of sodium salt components of oligonucleotides compared with LC-MS,and it also had a wide range of adaptability and sensitivity to detect a range of oligonucleotide molecules with different salt addition degrees.The method can also be used to analyze oligonucleotide sodium salt drugs(antisense oligonucleotide ASO,etc.).In this paper,the antisense oligonucleotide drug Formivesen sodium was taken as the sample,and the pure product of Formivesen sodium and the animal injection model of Formivesen sodium containing polyethylene glycol and Tween were tested by this method.The results showed that the main sodium addition form of Formivesen sodium ranged from 0 sodium

关 键 词：MALDI-TOF MS 寡核苷酸 寡核苷酸钠盐分布 定性分析 

分 类 号：O657.6[理学—分析化学] O629.74[理学—化学]