液相色谱-串联质谱分析化学合成多肽及其主要副产物(英文)

用与反相高效液相色谱偶联的串联质谱 (RPHPLC MS MS)分析一个固相化学合成的九肽H2 N Tyr Val Asn Val Asn Met Gly Leu Lys CONH2 及其 2个主要的副产物 .根据Fmoc化学 ,在Fmoc PAL PEG·PS树脂上从C端至N端手工操作逐步偶联合成九肽 .偶联完成后 ,用试剂R(90 %TFA ,5 %苯甲硫醚 ,3%二巯基乙烷 ,2 %苯甲醚 )室温 (2 0～ 2 5℃ )处理 2 5h ,进行多肽的去保护和切除树脂 .RP HPLC分析结果表明 ,合成粗品中含有 1个主要成分、2个次要成分及多个微量成分 .用RPHPLC MS MS分别对主要成分和 2个次要成分进行了鉴定 .结果证明 ,主要成分即为目标九肽 ;先于目标肽洗脱的副产物分子量比目标肽的分子量多 16 .MS MS证明 ,该副产物包括 2种九肽衍生物 :1种为其Tyr1氧化生成 ,另 1种为Met6氧化生成 ;后于目标肽洗脱的副产物为九肽的Tyr1残基增加 12所致 ,这种现象尚未见报道 .对副产物形成的可能机制进行了讨论

Liquid Chromatography-Tandem Mass Spectrometry Analysis of A Synthetic Nonapeptide and Its Main By-products

High performance liquid chromatography-tandem mass spectrometry was used to investigate a nonapeptide H2N-Tyr-Val-Asn-Val-Asn-Met-Gly-Leu-Lys-CONH2(Mr1035.6) and its main by-products from solid phase synthesis. The target sequence was assembled in stepwise fashion from the C-terminal to N-terminal using Fmoc chemistry on a Fmoc-PAL-PEG*PS resin manually. The product was deprotected and cleaved from the resin by treatment with reagent R (90%TFA, 5%thioanisole, 3%ethanedithiol, 2%anisole, V/V/V/V) for 2.5 hours at room temperature (20-25℃). Reverse-phase HPLC showed that the crude product contained a major component, two minor components (one early eluting and the other late eluting) and several trace-level components. The major component and the two minor components were analyzed by high performance liquid chromatography-tandem mass spectrometry, respectively. The major component was confirmed as the desired nonapeptide. The molecular weight of the early-eluting component was 16 more than that of the desired nonapeptide, and the by-product was proven to consist of two kinds of nonapeptide derivatives, one with Tyr1 oxidized and the other with Met6 oxidized. The late-eluting by-product was confirmed as resulting from 12 increase in mass of Tyr1 residue, which has not been reported early. The possible origins of those by-products were discussed.