作　　者： ; ; ; ; ; ; ;

机构地区： 广东省农业科学院

出　　处： 《华中师范大学学报（自然科学版）》 2018年第5期678-685,共8页

摘　　要： 为系统评估重组毕赤酵母植酸酶的N-糖基化充分性,建立了一种通过联用高效液相色谱-串级质谱来分析植酸酶氨基酸序列及N-糖基化位点的技术.分别发酵了3株基因组已整合不同植酸酶基因的毕赤酵母.发酵液上清经离子交换与凝胶过滤后获得纯化的植酸酶.使用PNGase F或Endo H脱糖基化酶处理植酸酶后,用胰蛋白酶消化脱糖基化产物.通过纳升级高效液相色谱分离胰蛋白酶切产生的肽段混合物.并在联用的串级质谱上进行肽段测序与N-糖基化位点鉴定.结果显示3种植酸酶蛋白的可检测序列覆盖率均在69%以上,且与文献报道不同,均存在糖基化位点修饰不充分的现象.本实验结果证明了高效液相色谱-串级质谱联用法检测植酸酶N-糖基化位点的有效性,并为植酸酶的糖基化研究提供了新的基础. To evaluate the sufficiency of the N-glycosylation of recombinant phytases from Pichia pastoris systematically,a method for analyzing the amino acid sequence and N-linked glycosylation sites of phytase was established via HPLC-MS/MS.Three strains of pichia pastoris,which have been recombined with different phytase genes,were fermented separately.The phytases were obtained by the sequential purification of ion-exchange and gel filtration.The purified phytases were treated by deglycosylases PNGase F or Endo H.Then the deglycosylated proteins were digested by trypsin.The tryptic mixtures were separated via Nano-HPLC.Peptide sequencing and site identification of N-linked glycosylation were performed on the coupled Tandem MS.The coverage for the detectable protein sequence is above 69%for every protein.Insufficient modifications of glycosylation sites have been observed in all phytases.The glycosylation patterns were different from that reported in the literature.The results demonstrated the efficiency of the detection of N-glycosylation sites in phytase via HPLC-MS/MS,and provide a new basis for the study of glycosylation of phytase.

关 键 词： 植酸酶 糖基化 质谱 毕赤酵母

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