重组人干扰素α1b质量肽图分析及二硫键定位

Peptide mass mapping analysis of rhIFN-α1b and localization of disulfide bonds

目的:液质联用绘制重组人干扰素α1b 的质量肽图并鉴定二硫键位点。方法:LC-MS 联用绘制重组人干扰素α1b 的胰蛋白酶酶切质量肽图;对比特定肽段的实测相对分子质量与理论相对分子质量初步定位二硫键,对比烷基化及还原烷基化处理后特定肽段实测相对分子质量的变化确证二硫键位点。结果:重组人干扰素α1b 的质谱测定相对分子质量为19382.50,与理论相对分子质量19382.18一致,其质量肽图中共发现16个匹配肽段,有3个理论酶切片段(单一氨基酸:Lys 135、Lys 165、Glu 166)未发现,氨基酸覆盖率为98.2%。通过分析胰蛋白酶酶切质量肽图,发现主要存在2种二硫键连接方式:Cys 29-Cys 139、Cys 86-Cys 99,同时还存在少量其他二硫键连接方式,如:C1-C99。结论:质量肽图可作为一种对重组蛋白制品进行质量控制的手段,并可定位蛋白中的二硫键。

Objective： To measure peptide mass mapping （PMM） of rhlFN - αlb by LC - MS and identify the sites of disulfide bonds. Methods：The PMM of rhlFN - αLb digested by trypsin was measured by LC - MS;the sites of disulfide bonds were identified roughly by contrasting measured relative molecular mass with theoretical value of specific peptides, and it was confirmed by specific relative molecular mass variations between peptides alkylated and hydrogenized before alkylated. Results： The measured relative molecular mass of rhlFN -αLb was 19382. 50 and the theoretical value is 19382. 18.16 Matched peptides were found in the PMM of rhlFN -αLb digested by trypsin, and 3 theoretically digested peptides（single amino acides：Lys 135 ,Lys 165,Glu 166） were not found,the fraction of coverage was 98.2%. The PMM of rhlFN - otlb digested by trypsin showed that the major types of disulfide bonds existing in protein were Cys 29 - Cys 139 and Cys 86 - Cys 99. Other types in small amounts also existed such as C1 - C99. Conclusion： PMM can be a method for the quality control of recombination protein, and it can lo- calize the sites of disulfide bonds.