Huber C G, Krajete A
Institute of Analytical Chemistry and Radiochemistry, Leopold Franzens University, Innsbruck, Austria.
J Mass Spectrom. 2000 Jul;35(7):870-7. doi: 10.1002/1096-9888(200007)35:7<870::AID-JMS11>3.0.CO;2-D.
The applicability of ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) and direct infusion/ESI-MS to the characterization of nucleic acid mixtures was evaluated by the analysis of the reaction products obtained from solid-phase synthesis of a 39-mer oligonucleotide. IP-RP-HPLC/ESI-MS was performed using 200 microm i.d. capillary columns packed with octadecylated, micropellicular poly(styrene-divinylbenzene) particles and applying gradients of acetonitrile in 50 mM triethylammonium bicarbonate (TEAB). Three different solvent systems were utilized for direct infusion/ESI-MS with removal of metal cations by on-line cation exchange: (1) 10 mM triethylamine (TEA) in 50% aqueous acetonitrile, (2) 2.2 mM TEA, 400 mM hexafluoro-2-propanol (HFIP) in 20% aqueous methanol and (3) 50 mM TEAB in 10% aqueous acetonitrile. Owing to its separation capability, the highest selectivity and specificity were achieved with IP-RP-HPLC/ESI-MS, which, apart form the 39-mer target sequence, allowed the identification of two isobutyryl-protected target sequences and a 10-mer and 20-mer failure sequence. Direct infusion/ESI-MS with TEA-acetonitrile or TEA-HFIP-methanol as solvent revealed signals for the 39-mer in the m/z range 700-1600. The presence of derivatives containing one, two, three and four isobutyryl groups indicated that the hydrolysis of the protecting groups after solid-phase synthesis was not complete. Failure sequences could not be identified by direct infusion/ESI-MS under conditions favoring multiple charging of the analytes owing to the high chemical background and coincidental overlapping of m/z signals. However, efficient charge state reduction upon addition of carbonic acid to the electrosprayed solvent shifted the signals of the 39-mer and derivatives to m/z values >2400 and allowed the detection of seven different failure sequences, ranging from the 8-mer to the 23-mer, in the mixture.
通过对固相合成39聚体寡核苷酸所得反应产物的分析，评估了离子对反相高效液相色谱/电喷雾电离质谱法（IP-RP-HPLC/ESI-MS）和直接进样/ESI-MS在核酸混合物表征中的适用性。IP-RP-HPLC/ESI-MS采用内径200微米的毛细管柱，填充十八烷基化的微孔聚（苯乙烯-二乙烯基苯）颗粒，并在50 mM碳酸氢三乙胺（TEAB）中应用乙腈梯度。采用三种不同的溶剂体系进行直接进样/ESI-MS，并通过在线阳离子交换去除金属阳离子：（1）50%乙腈水溶液中的10 mM三乙胺（TEA），（2）20%甲醇水溶液中的2.2 mM TEA、400 mM六氟-2-丙醇（HFIP），（3）10%乙腈水溶液中的50 mM TEAB。由于其分离能力，IP-RP-HPLC/ESI-MS实现了最高的选择性和特异性，除了39聚体目标序列外，还能鉴定出两个异丁酰基保护的目标序列以及一个10聚体和一个20聚体失败序列。以TEA-乙腈或TEA-HFIP-甲醇为溶剂的直接进样/ESI-MS在m/z范围700-1600内显示出39聚体的信号。含有一个、两个、三个和四个异丁酰基的衍生物的存在表明固相合成后保护基团的水解不完全。由于高化学背景和m/z信号的巧合重叠，在有利于分析物多重充电的条件下，直接进样/ESI-MS无法鉴定失败序列。然而，在电喷雾溶剂中加入碳酸后有效降低电荷态，将39聚体及其衍生物的信号转移到m/z值>2400，并允许检测混合物中七个不同的失败序列，范围从8聚体到23聚体。
