2021 04 v.32;No.104 439-445+459
基于特征肽段定量新冠病毒刺突蛋白S1亚基的方法建立
基金项目(Foundation):
国家市场监督管理总局技术保障项目(No.2020YJ058);;
浙江省自然科学基金项目(No.LGC21C010001)
邮箱(Email):
zhye@cjlu.edu.cn;
DOI:
中文作者单位:
中国计量大学生命科学学院;
摘要(Abstract):
目的:建立基于特征肽段的新冠病毒刺突蛋白S1亚基定量方法。方法:根据蛋白序列、糖基化修饰位点等筛选特征肽段;利用胰蛋白酶水解蛋白为相对分子质量较小的肽段,并优化反应参数;最后结合同位素稀释质谱法建立刺突蛋白S1亚基定量检测方法,并进行方法学考察。结果:基于特征肽段的同位素稀释质谱法检测样品中S1亚基质量分数为1.007×10~(-3),扩展不确定度为0.070×10~(-3),结果准确,与基于氨基酸分析的同位素稀释质谱法检测结果比较无显著差异。特征肽段质量分数在0.47×10~(-9)～1×10~(-3)范围内,回归相关系数R~2>0.999 9,线性良好。该方法日内与日间精密度均小于5%,检测准确度均在95%～105%之间,重复性良好。该方法不受基质中其他蛋白的干扰,可溯源至国际单位制,有助于该蛋白绝对定量、标准物质研制等。结论:该方法可行性良好,后期可供参考用于新冠病毒刺突蛋白S1亚基的定量。
关键词(KeyWords):
新型冠状病毒;;刺突蛋白S1亚基;;特征肽段;;定量方法;;同位素稀释质谱法
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[17] 高颖，杨怡，欧阳艳艳，等.新型冠状病毒标准物质研究进展[J].中华实验和临床病毒学杂志，2021,35(5):590-595.GAO Y,YANG Y,OUYANG Y Y,et al.Research progress in reference materials for new coronaviruses[J].Chinese Journal of Experimental and Clinical Virology,2021,35(5):590-595.
[2] YIN Y D,WUNDERINK R G.MERS,SARS and other coronaviruses as causes of pneumonia[J].Respirology (Carlton,Vic.),2018,23(2):130-137.
[3] 王越珉，雷喜梅，邬丽，等.新型冠状病毒及其检测方法研究进展[J].中国计量大学学报，2020,31(1):1-7.WANG Y M,LEI X M,WU L,et al.A review of severe acute respiratory syndrome coronavirus 2 and its detecting methods[J].Journal of China University of Metrology,2020,31(1):1-7.
[4] 王淑燕，李娜，张欣悦，等.新冠病毒SARS-CoV-2检测方法研究进展[J].实验室研究与探索，2020,39(5):1-7,16.WANG S Y,LI N,ZHANG X Y,et al.Recent advances on detection methods of novel coronavirus SARS-CoV-2[J].Research and Exploration in Laboratory,2020,39(5):1-7,16.
[5] LI F.Structure,function,and evolution of coronavirus spike proteins[J].Annual Review of Virology,2016,3(1):1954-1964.
[6] 朱金蕾，张锴，何锡文，等.基于质谱技术蛋白质定量方法的研究进展[J].分析化学，2010,38(3):434-441.ZHU J L,ZHANG K,HE X W,et al.Research progress of protein quantification methods based on mass spectrometry[J].Chinese Journal of Analytical Chemistry,2010,38(3):434-441.
[7] 韩宁娟，方欢乐，刘建利.生物样品中多肽定量分析方法[J].生物化工，2021,7(1):157-159.HAN N J,FANG H,LIU J L.Research progress on quantitative analysis methods of peptides in biological samples[J].Biological Chemical Engineering,2021,7(1):157-159.
[8] 孙雪晴.重要生物标志物—甲胎蛋白、瘦素及肌钙蛋白Ⅰ的绝对定量方法[D].北京：北京化工大学，2014:46-49.SUN X Q.Absolute Quantification of the Important Biomarkers—Alpha-fetoprotein,Leptin and Recombinant Cardiac Troponin Through Isotope Dilution Mass Spectrometry[D].Beijing:Beijing University of Chemical Technology,2014:46-49.
[9] 王群利，陈晓红.同位素内标稀释-分散固相萃取-液相色谱串联质谱测定环境水中五氯酚[J].中国卫生检验杂志，2020,30(7):775-778,782.WANG Q L,CHEN X H.Determination of pentachlorophenol in water by isotope internal standard dilution—dispersive solid phase extraction—liquid chromatography coupled with tandem mass spectrometry[J].Chinese Journal of Health Laboratory Technology,2020,30(7):775-778,782.
[10] 孙贺伟，居漪，朱岭峰，等.同位素稀释液相色谱-串联质谱法检测血清睾酮候选参考方法的建立及应用[J].检验医学，2021,36(10):1064-1069.SUN H W,JU Y,ZHU L F,et al.Isotope dilution liquid chromatography tandem mass spectrometry candidate reference method for serum testosterone[J].Laboratory Medicine,2021,36(10):1064-1069.
[11] WHITING G,FERGUSON J,FANG M,et al.Quantification of müllerian inhibiting substance/anti-müllerian hormone polypeptide by isotope dilution mass spectrometry[J].Analytical Biochemistry,2018,560:50-55.
[12] 张宏康，王中瑗，杨启津，等.同位素稀释质谱法在食品分析中的应用研究进展[J].食品科学，2018,39(23):280-288.ZHANG H K,WANG Z Y,YANG Q J,et al.Recent advances in application of isotopic dilution mass spectrometry in food analysis[J].Food Science,2018,39(23):280-288.
[13] 刘钰，宋德伟，张方彦，等.甲胎蛋白纯品的同位素稀释质谱法测定[J].质谱学报，2017,38(6):640-646.LIU Y,SONG D W,ZHANG F Y,et al.Measurement of alpha-fetoprotein by isotope dilution mass spectrometry[J].Journal of Chinese Mass Spectrometry Society,2017,38(6):640-646.
[14] 宋德伟，董晓杰，徐蓓，等.C-反应蛋白纯品的同位素稀释质谱法测定与国际比对[J].质谱学报，2014,35(5):462-466.SONG D W,DONG X J,XU B,et al.Measurement and internationalcomparison for C-reactive protein by isotope dilution mass spectrometry[J].Journal of Chinese Mass Spectrometry Societ,2014,35(5):462-466.
[15] 沈枫林，熊悦婷，刘晓慧，等.基于质谱的蛋白质绝对定量研究策略和建议[J].质谱学报，2021,42(2):101-117.SHEN F L,XIONG Y T,LIU X H,et al.Strategies and recommendations for the absolute quantifition of target proteins based on LC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2021,42(2):101-117.
[16] 王雪颖，秦伟捷，钱小红，等.氨基酸-稳定同位素稀释质谱法用于合成肽段准确含量分析[J].色谱，2012,30(3):239-244.WANG X Y,QIN W J,QIAN X H,et al.Accurate quantification of synthetic peptides by amino acid-stable isotope dilution mass spectrometry[J].Chinese Journal of Chromatography,2012,30(3):239-244.
[17] 高颖，杨怡，欧阳艳艳，等.新型冠状病毒标准物质研究进展[J].中华实验和临床病毒学杂志，2021,35(5):590-595.GAO Y,YANG Y,OUYANG Y Y,et al.Research progress in reference materials for new coronaviruses[J].Chinese Journal of Experimental and Clinical Virology,2021,35(5):590-595.
基本信息:
DOI：
中图分类号:R373
引用信息:
[1]马玉清,夏文强,张雅芬等.基于特征肽段定量新冠病毒刺突蛋白S1亚基的方法建立[J].中国计量大学学报,2021,32(04):439-445+459.
基金信息:
国家市场监督管理总局技术保障项目(No.2020YJ058);; 浙江省自然科学基金项目(No.LGC21C010001)
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