目的: 使用超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q TOF MS法)对国内外3家企业溶菌酶的一级结构进行分析研究。方法: 采用BEH300 C4(50 mm×2.1 mm, 1.7 μm)色谱柱和BEH300 C18(150 mm×2.1 mm, 1.7 μm)色谱柱,以0.1%甲酸/水溶液为流动相A,0.1%甲酸/乙腈溶液为流动相B,梯度洗脱,流速0.2 mL·min-1;采用Q TOF MS的电喷雾正离子模式,相对分子质量测定时采用MS模式进行扫描,氨基酸序列和二硫键连接方式测定时采用MSE模式进行扫描。结果: 3种来源溶菌酶相对分子质量与理论值一致;肽图结果显示各样品的氨基酸序列均与理论一致;二硫键连接方式均为Cys6-Cys127、Cys30-Cys115、Cys64-Cys80、Cys76-Cys94。结论: UPLC-Q TOF MS法可用于溶菌酶一级结构的分析。
Objective: To analyze the primary structure of the lysozyme from three different domestic and foreign manufacturers using UPLC-Q TOF MS. Methods: The UPLC-Q TOF MS analysis was performed on a BEH300 C4(50 mm×2.1 mm, 1.7 μm) column and a BEH300 C18(150 mm×2.1 mm, 1.7 μm) column with 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid-acetonitrile solution as mobile phase B, with gradient elution at a flow rate of 0.2 mL·min-1. Q TOF MS electrospray positive ion mode was used, and for relative molecular mass detection, MS scan mode was applied. For amino acid sequence and disulfide bond connection, MSE scan mode was applied. Results: The measured relative molecular mass of the three lysozyme samples was consistent with theory; the results of peptide map showed that the amino acid sequences of all samples were consistent with the theory; the connection modes of disulfide bonds of all lysozymes were Cys6-Cys127, Cys30-Cys115,Cys64-Cys80 and Cys76-Cys94. Conclusion: UPLC-Q TOF MS method can be used to analyze the primary structure of the lysozyme.
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