探讨Lys-C胰蛋白酶对于质谱法测定糖化血红蛋白的应用价值

Application value of Lys-C trypsin for the detection of HbA1c by using mass spectrometric method*

目的探讨Lys-C胰蛋白酶在国际临床化学组织(International Federation of Clinical Chemistry and Laboratory Medicine,IFCC)推荐的质谱法测定糖化血红蛋白的参考方法中的应用价值。方法分别用两种特异性蛋白水解酶(GLU-C胞内蛋白酶和Lys-C胰蛋白酶)酶解血红蛋白后,进行SDS-PAGE电泳,衡量蛋白酶解的效率,再将消化后的血红蛋白进行质谱分析,测定标本中糖化血红蛋白的浓度。结果在相同的电泳条件下,Lys-C胰蛋白酶酶解血红蛋白的效率优于GLU-C;酶解后的血红蛋白标本经质谱方法分析可知,两种蛋白酶用于检测糖化血红蛋白浓度时,可以得到基本一致的结果,由两种方法绘制的标准曲线r2分别为0.929和0.998。结论在IFCC推荐的糖化血红的蛋白参考测量方法中,一直使用GLU-C胞内蛋白酶酶解血红蛋白,该研究尝试通过Lys-C胰蛋白酶的使用,降低整个实验成本。经结果分析,证实应用这两种特异性的蛋白酶检测糖化血红蛋白时,可以得到基本一致的结果,推荐使用文中所列方法将Lys-C胰蛋白酶作为酶解糖化血红蛋白的特异性蛋白酶。

Objective To explore the application value of Lys-C trypsin for the detection of glycosylated hemoglobin A1 （HbAlc） using the mass spectrometric method as reference method recommended by International Federation of Clinical Chemistry and Laboratory Medicine（IFCC）. Methods Hemoglobin was enzymatically hydrolyzed by two kinds of proteolytic enzymes, inclu- ding intracel｜ular protease GLU-C and Lys-C trypsin. SDS-PAGE was performed to measure the enzymatic hydrolysis efficiency, and after digestion of hemoglobin, mass spectrometry was used to determinate the HbAlc concentration. Results According to SDS-PAGE results, under the same electrophoresis conditions, the hydrolysis efficiency of Lys-C trypsin higher than GLU-C. Ac- cording to LTQ-Orbitrap Velos mass spectrometer analysis results, HbAlc concentrations in specimens treated with the two kinds of protease mitht be basically consistent, and r2 values of the two standard curves of the two methods were 0. 929 and 0. 998 re- spectively. Conclusion Treatment of Lys-C trypsin and GLU-C could acquire the same detection resulsts of HbAlc detected by ref- erence method recommended by IFCC, and Lys-C trypsin could replacing GLU--C for the detection of HbAlc.