基于纳升电喷雾离子化质谱技术(nano ESI-MS)建立登革病毒检测方法
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摘要
目的建立基于纳升电喷雾离子化质谱技术(nano ESI-MS)检测登革病毒的方法。方法采用生物工程技术,合成Ⅱ型登革病毒(DENV-Ⅱ)NS1蛋白特异性肽段作为识别标志,通过优化实验条件,建立基于nano ESI-MS技术的DENV-ⅡNS1蛋白特异性肽段的检测方法;借助特异性胰蛋白酶酶解技术,实现对DENV-ⅡNS1蛋白的检测验证。结果在2.5 kV电压、以甲醇、水按体积比6∶4比例并加入0.1%甲酸作为蛋白稀释液条件下,应用nano ESI-MS技术可成功检测出DENV-ⅡNS1蛋白特异性标志性肽段,灵敏度达10 ng/ml。结论 nano ESI-MS技术灵敏度高,操作简单,可在敞开式大气压环境下实现检测。该方法的建立为扩展nano ESI-MS技术应用范围提供了新思路。
Objective To develop a method based on nano-electrospray ionization spectrometry(nano ESI-MS)technique to test Dengue virus(DENV).Methods The specific peptide of DENV-ⅡNS1 protein expressed by bioengineering technology was used as an identified marker.The detection based on nano ESI-MS was established to test DENV-ⅡNS1 protein by specific trypsin hydrolysis technology.Results Under the condition of 2.5kV,methanol:water was 6:4(by volume)with 0.1%formic acid as the diluent of protein,the nano ESI-MS method had set up to detect the specific peptide of DENV-ⅡNS1 protein.The limitation was 10 ng/ml.Conclusion nano ESI-MS method was sensitive with simple operation,and can be used to run at atmospheric pressure.The establishment of this method is expected to provide a new idea for the detection of infectious disease pathogens.
Objective To develop a method based on nano-electrospray ionization spectrometry(nano ESI-MS)technique to test Dengue virus(DENV).Methods The specific peptide of DENV-ⅡNS1 protein expressed by bioengineering technology was used as an identified marker.The detection based on nano ESI-MS was established to test DENV-ⅡNS1 protein by specific trypsin hydrolysis technology.Results Under the condition of 2.5kV,methanol:water was 6:4(by volume)with 0.1%formic acid as the diluent of protein,the nano ESI-MS method had set up to detect the specific peptide of DENV-ⅡNS1 protein.The limitation was 10 ng/ml.Conclusion nano ESI-MS method was sensitive with simple operation,and can be used to run at atmospheric pressure.The establishment of this method is expected to provide a new idea for the detection of infectious disease pathogens.
引文
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[15] Danne-Rasche N,Coman C,Ahrends R. Nano-LC/NSI MS refines lipidomics by enhancing lipid coverage,measurement sensitivity,and linear dynamic range[J]. Anal Chem,2018,90(13):8093-8101.
[16] Maes K,Béchade G,Van Schoors,et al. An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates[J]. Bioanalysis,2015,7(5):605-619.
[17] Zhang Z,Pulliam CJ,Flick T,Cooks RG,et al. Electrophoretic desalting to improve performance in electrospray ionization mass spectrometry[J]. Anal Chem,2018,90(6):3856-3862.
[2] Hu TS,Zhang HL,Feng Y,et al. Epidemiological and molecular characteristics of emergent dengue virus in Yunnan Province near the China-Myanmar-Laos border,2013-2015[J]. BMC Infect Dis,2017,17(1):331-343.
[3]林钟宇,潘华峰,王正,等. 2014年广东登革热流行趋势与防控对策[J].卫生软科学,2015,29(09):590-592.
[4] Saito Y,Moi ML,Kotaki A,et al. Detecting Dengue virus nonstructural protein 1(ns1)in urine samples using ELISA for the diagnosis of dengue virus infection[J]. Jpn J Infect Dis,2015,68(6):455-460.
[5] Alcon S,Talarmin A,Debruyne M,et al. Enzyme-linked immunosorbent assay specific to Dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections[J]. J Clin Microbiol,2002,40(2):376-381.
[6] Vasanwala FF,Thein TL,Leo YS,et al. Predictive value of proteinuria in adult dengue severity[J]. PLoS Negl Trop Dis,2014,8(2):e2712.
[7] Calderaro A,Arcangeletti MC,Rodighiero,et al. Matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF)mass spectrometry applied to virus identification[J]. Scientific Reports,2014(4):6803.
[8] Chou TC,Hsu W,Wang CH,et al. Rapid and specific influenza virus detection by functionnalized magnetic nanoparticles and mass spectrometry[J]. Journal of nanobiotechnology,2011,9(1):52-65.
[9] Brady OJ,Gething PW,Bhatt S,et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus[J]. PLoS Negl Trop Dis,2012,6(8):e1760.
[10] Woolman M,Zarrine-Afsar A. Platforms for rapid cancer characterization by ambient mass spectrometry:advancements,challenges and opportunities for improvement towards intrasurgical use[J]. Analyst,2018,143(12):2717-2722.
[11] Banerjee S,Mazumdar S. Electrospray ionization mass spectrometry:a technique to access the information beyond the molecular weight of the analyte[J]. Int J Anal Chem,2012.doi:10.155/2012/282574.
[12] Zhang M,Lin F,Xu J,et al. Membrane electrospray ionization for direct ultrasensitive biomarker quantitation in biofluids using mass spectrometry[J]. Anal Chem,2015,87(6):3123-3128.
[13]许光明.快速表面质谱分析方法及其应用研究[D].长沙:湖南大学,2013.
[14] Wilm M,Mann M. Analytical properties of the nanoelectrospray ion source[J]. Anal Chem,1996,68(1):1-8.
[15] Danne-Rasche N,Coman C,Ahrends R. Nano-LC/NSI MS refines lipidomics by enhancing lipid coverage,measurement sensitivity,and linear dynamic range[J]. Anal Chem,2018,90(13):8093-8101.
[16] Maes K,Béchade G,Van Schoors,et al. An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates[J]. Bioanalysis,2015,7(5):605-619.
[17] Zhang Z,Pulliam CJ,Flick T,Cooks RG,et al. Electrophoretic desalting to improve performance in electrospray ionization mass spectrometry[J]. Anal Chem,2018,90(6):3856-3862.